US20110023149A1 - Genomic editing of genes involved in tumor suppression in animals - Google Patents
Genomic editing of genes involved in tumor suppression in animals Download PDFInfo
- Publication number
- US20110023149A1 US20110023149A1 US12/842,978 US84297810A US2011023149A1 US 20110023149 A1 US20110023149 A1 US 20110023149A1 US 84297810 A US84297810 A US 84297810A US 2011023149 A1 US2011023149 A1 US 2011023149A1
- Authority
- US
- United States
- Prior art keywords
- receptor
- protein
- sequence
- growth factor
- caspase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 147
- 230000005760 tumorsuppression Effects 0.000 title claims abstract description 112
- 108090000623 proteins and genes Proteins 0.000 title claims description 200
- 230000002759 chromosomal effect Effects 0.000 claims abstract description 140
- 108010017070 Zinc Finger Nucleases Proteins 0.000 claims abstract description 77
- 238000000034 method Methods 0.000 claims abstract description 58
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 53
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 158
- 210000004027 cell Anatomy 0.000 claims description 134
- 102000040430 polynucleotide Human genes 0.000 claims description 74
- 108091033319 polynucleotide Proteins 0.000 claims description 74
- 239000002157 polynucleotide Substances 0.000 claims description 74
- 206010028980 Neoplasm Diseases 0.000 claims description 54
- 238000003776 cleavage reaction Methods 0.000 claims description 52
- 230000007017 scission Effects 0.000 claims description 52
- 101000859758 Homo sapiens Cartilage-associated protein Proteins 0.000 claims description 48
- 101000916686 Homo sapiens Cytohesin-interacting protein Proteins 0.000 claims description 48
- 101000726740 Homo sapiens Homeobox protein cut-like 1 Proteins 0.000 claims description 48
- 101000761460 Homo sapiens Protein CASP Proteins 0.000 claims description 48
- 101000761459 Mesocricetus auratus Calcium-dependent serine proteinase Proteins 0.000 claims description 48
- 102000005962 receptors Human genes 0.000 claims description 47
- 108020003175 receptors Proteins 0.000 claims description 47
- 210000001161 mammalian embryo Anatomy 0.000 claims description 43
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims description 37
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 35
- 239000011701 zinc Substances 0.000 claims description 35
- 229910052725 zinc Inorganic materials 0.000 claims description 35
- 108700020796 Oncogene Proteins 0.000 claims description 34
- 241000282414 Homo sapiens Species 0.000 claims description 33
- 230000003612 virological effect Effects 0.000 claims description 33
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 claims description 30
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 claims description 27
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 claims description 27
- 101000785063 Homo sapiens Serine-protein kinase ATM Proteins 0.000 claims description 25
- 102000036365 BRCA1 Human genes 0.000 claims description 24
- 108700020463 BRCA1 Proteins 0.000 claims description 24
- 102000000872 ATM Human genes 0.000 claims description 23
- 206010006187 Breast cancer Diseases 0.000 claims description 23
- 208000026310 Breast neoplasm Diseases 0.000 claims description 23
- 108010009392 Cyclin-Dependent Kinase Inhibitor p16 Proteins 0.000 claims description 23
- 108700031765 Von Hippel-Lindau Tumor Suppressor Proteins 0.000 claims description 23
- 208000021841 acute erythroid leukemia Diseases 0.000 claims description 23
- 230000014509 gene expression Effects 0.000 claims description 23
- 102000052609 BRCA2 Human genes 0.000 claims description 22
- 108700020462 BRCA2 Proteins 0.000 claims description 22
- 102100032187 Androgen receptor Human genes 0.000 claims description 21
- 108010080146 androgen receptors Proteins 0.000 claims description 21
- 102000001301 EGF receptor Human genes 0.000 claims description 20
- 108060006698 EGF receptor Proteins 0.000 claims description 20
- 102100027619 Histidine-rich glycoprotein Human genes 0.000 claims description 20
- 108010044853 histidine-rich proteins Proteins 0.000 claims description 20
- 102100037182 Cation-independent mannose-6-phosphate receptor Human genes 0.000 claims description 19
- 101710145225 Cation-independent mannose-6-phosphate receptor Proteins 0.000 claims description 19
- 102100039688 Insulin-like growth factor 1 receptor Human genes 0.000 claims description 19
- 101710184277 Insulin-like growth factor 1 receptor Proteins 0.000 claims description 19
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 claims description 18
- 206010073150 Multiple endocrine neoplasia Type 1 Diseases 0.000 claims description 18
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 claims description 18
- 108700000707 bcl-2-Associated X Proteins 0.000 claims description 18
- 102000055102 bcl-2-Associated X Human genes 0.000 claims description 18
- 101000779418 Homo sapiens RAC-alpha serine/threonine-protein kinase Proteins 0.000 claims description 17
- 101000904787 Homo sapiens Serine/threonine-protein kinase ATR Proteins 0.000 claims description 17
- 102100023181 Neurogenic locus notch homolog protein 1 Human genes 0.000 claims description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 17
- 102100023921 Serine/threonine-protein kinase ATR Human genes 0.000 claims description 17
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 claims description 17
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 16
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 16
- 102100026548 Caspase-8 Human genes 0.000 claims description 15
- 206010021143 Hypoxia Diseases 0.000 claims description 14
- 101000798015 Homo sapiens RAC-beta serine/threonine-protein kinase Proteins 0.000 claims description 13
- 101000798007 Homo sapiens RAC-gamma serine/threonine-protein kinase Proteins 0.000 claims description 13
- 101150002416 Igf2 gene Proteins 0.000 claims description 13
- 101150014691 PPARA gene Proteins 0.000 claims description 13
- 101710100969 Receptor tyrosine-protein kinase erbB-3 Proteins 0.000 claims description 13
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 claims description 12
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 claims description 12
- 108010011536 PTEN Phosphohydrolase Proteins 0.000 claims description 12
- 102100032315 RAC-beta serine/threonine-protein kinase Human genes 0.000 claims description 12
- 102100029986 Receptor tyrosine-protein kinase erbB-3 Human genes 0.000 claims description 12
- 229940029303 fibroblast growth factor-1 Drugs 0.000 claims description 12
- 101150088952 IGF1 gene Proteins 0.000 claims description 11
- 102100032314 RAC-gamma serine/threonine-protein kinase Human genes 0.000 claims description 11
- 239000000225 tumor suppressor protein Substances 0.000 claims description 11
- 208000029664 classic familial adenomatous polyposis Diseases 0.000 claims description 10
- 101000742859 Homo sapiens Retinoblastoma-associated protein Proteins 0.000 claims description 9
- 108010029741 Notch4 Receptor Proteins 0.000 claims description 9
- 102100029981 Receptor tyrosine-protein kinase erbB-4 Human genes 0.000 claims description 9
- 101710100963 Receptor tyrosine-protein kinase erbB-4 Proteins 0.000 claims description 9
- 102100038042 Retinoblastoma-associated protein Human genes 0.000 claims description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 8
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 claims description 8
- 101001076292 Homo sapiens Insulin-like growth factor II Proteins 0.000 claims description 8
- 102100037852 Insulin-like growth factor I Human genes 0.000 claims description 8
- 102100025947 Insulin-like growth factor II Human genes 0.000 claims description 8
- 108010016731 PPAR gamma Proteins 0.000 claims description 8
- 102100038825 Peroxisome proliferator-activated receptor gamma Human genes 0.000 claims description 8
- 206010039491 Sarcoma Diseases 0.000 claims description 8
- 229940068935 insulin-like growth factor 2 Drugs 0.000 claims description 8
- 108090000397 Caspase 3 Proteins 0.000 claims description 7
- 102100035904 Caspase-1 Human genes 0.000 claims description 7
- 102100029855 Caspase-3 Human genes 0.000 claims description 7
- 102100025597 Caspase-4 Human genes 0.000 claims description 7
- 102100038902 Caspase-7 Human genes 0.000 claims description 7
- 108090000538 Caspase-8 Proteins 0.000 claims description 7
- 230000004568 DNA-binding Effects 0.000 claims description 7
- 229910019142 PO4 Inorganic materials 0.000 claims description 7
- 102000003728 Peroxisome Proliferator-Activated Receptors Human genes 0.000 claims description 7
- 108090000029 Peroxisome Proliferator-Activated Receptors Proteins 0.000 claims description 7
- 102100038831 Peroxisome proliferator-activated receptor alpha Human genes 0.000 claims description 7
- 101710173511 Tensin homolog Proteins 0.000 claims description 7
- 108091008725 peroxisome proliferator-activated receptors alpha Proteins 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 7
- 239000010452 phosphate Substances 0.000 claims description 7
- 230000004044 response Effects 0.000 claims description 7
- 101100181122 Arabidopsis thaliana KIN14C gene Proteins 0.000 claims description 6
- 101100234530 Arabidopsis thaliana KIN14M gene Proteins 0.000 claims description 6
- 101100234532 Arabidopsis thaliana KIN14N gene Proteins 0.000 claims description 6
- 101100288145 Arabidopsis thaliana KNAT2 gene Proteins 0.000 claims description 6
- 108090000425 Caspase 6 Proteins 0.000 claims description 6
- 108090000567 Caspase 7 Proteins 0.000 claims description 6
- 108090000426 Caspase-1 Proteins 0.000 claims description 6
- 108090000570 Caspase-12 Proteins 0.000 claims description 6
- 102000004066 Caspase-12 Human genes 0.000 claims description 6
- 108090000552 Caspase-2 Proteins 0.000 claims description 6
- 101710090338 Caspase-4 Proteins 0.000 claims description 6
- 108090000566 Caspase-9 Proteins 0.000 claims description 6
- 102000003975 Fibroblast growth factor 3 Human genes 0.000 claims description 6
- 108090000378 Fibroblast growth factor 3 Proteins 0.000 claims description 6
- 102000003969 Fibroblast growth factor 4 Human genes 0.000 claims description 6
- 108090000381 Fibroblast growth factor 4 Proteins 0.000 claims description 6
- 102000003967 Fibroblast growth factor 5 Human genes 0.000 claims description 6
- 108090000380 Fibroblast growth factor 5 Proteins 0.000 claims description 6
- 241000283984 Rodentia Species 0.000 claims description 5
- 231100000419 toxicity Toxicity 0.000 claims description 5
- 230000001988 toxicity Effects 0.000 claims description 5
- 241000283690 Bos taurus Species 0.000 claims description 4
- 241000282465 Canis Species 0.000 claims description 4
- 230000001225 therapeutic effect Effects 0.000 claims description 4
- 230000000366 juvenile effect Effects 0.000 claims description 2
- 102100024933 Protein CASP Human genes 0.000 claims 24
- 102100033254 Tumor suppressor ARF Human genes 0.000 claims 6
- 102100035070 von Hippel-Lindau disease tumor suppressor Human genes 0.000 claims 6
- 102000014160 PTEN Phosphohydrolase Human genes 0.000 claims 4
- 102000004018 Caspase 6 Human genes 0.000 claims 3
- 102000004046 Caspase-2 Human genes 0.000 claims 3
- 102000004039 Caspase-9 Human genes 0.000 claims 3
- 241000283073 Equus caballus Species 0.000 claims 3
- 241000282324 Felis Species 0.000 claims 3
- 102000039446 nucleic acids Human genes 0.000 abstract description 38
- 108020004707 nucleic acids Proteins 0.000 abstract description 38
- 230000000694 effects Effects 0.000 abstract description 18
- 230000008569 process Effects 0.000 abstract description 18
- 230000001404 mediated effect Effects 0.000 abstract description 12
- 235000018102 proteins Nutrition 0.000 description 138
- 230000035772 mutation Effects 0.000 description 89
- 239000002773 nucleotide Substances 0.000 description 43
- 125000003729 nucleotide group Chemical group 0.000 description 42
- 108020004414 DNA Proteins 0.000 description 39
- 238000012360 testing method Methods 0.000 description 32
- 230000027455 binding Effects 0.000 description 30
- 241000700159 Rattus Species 0.000 description 26
- 102100030234 Homeobox protein cut-like 1 Human genes 0.000 description 24
- 238000009396 hybridization Methods 0.000 description 23
- 239000000178 monomer Substances 0.000 description 22
- 201000011510 cancer Diseases 0.000 description 18
- 238000012217 deletion Methods 0.000 description 18
- 230000037430 deletion Effects 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 17
- 102000053200 Von Hippel-Lindau Tumor Suppressor Human genes 0.000 description 17
- 229940088598 enzyme Drugs 0.000 description 17
- 102100024458 Cyclin-dependent kinase inhibitor 2A Human genes 0.000 description 16
- 230000002068 genetic effect Effects 0.000 description 16
- 238000011144 upstream manufacturing Methods 0.000 description 16
- 230000001965 increasing effect Effects 0.000 description 15
- 230000010354 integration Effects 0.000 description 15
- 108020004999 messenger RNA Proteins 0.000 description 15
- 108700037638 Neurogenic locus notch homolog protein 1 Proteins 0.000 description 13
- 108010029756 Notch3 Receptor Proteins 0.000 description 13
- 229940024606 amino acid Drugs 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 13
- 230000032823 cell division Effects 0.000 description 13
- 102100025246 Neurogenic locus notch homolog protein 2 Human genes 0.000 description 12
- 108700037064 Neurogenic locus notch homolog protein 2 Proteins 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 12
- 230000018109 developmental process Effects 0.000 description 12
- 230000008439 repair process Effects 0.000 description 12
- 238000011161 development Methods 0.000 description 11
- 125000003275 alpha amino acid group Chemical group 0.000 description 10
- 239000002246 antineoplastic agent Substances 0.000 description 10
- 230000006907 apoptotic process Effects 0.000 description 10
- 229940127089 cytotoxic agent Drugs 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 10
- 230000006870 function Effects 0.000 description 10
- 206010061535 Ovarian neoplasm Diseases 0.000 description 9
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 9
- 238000003780 insertion Methods 0.000 description 9
- 230000037431 insertion Effects 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- 208000011580 syndromic disease Diseases 0.000 description 9
- 101150065175 Atm gene Proteins 0.000 description 8
- 101001046870 Homo sapiens Hypoxia-inducible factor 1-alpha Proteins 0.000 description 8
- 102100022875 Hypoxia-inducible factor 1-alpha Human genes 0.000 description 8
- 102100025247 Neurogenic locus notch homolog protein 3 Human genes 0.000 description 8
- 102100032543 Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN Human genes 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 8
- 230000022131 cell cycle Effects 0.000 description 8
- 210000002257 embryonic structure Anatomy 0.000 description 8
- 208000020816 lung neoplasm Diseases 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- 241000271566 Aves Species 0.000 description 7
- 206010009944 Colon cancer Diseases 0.000 description 7
- 101150105104 Kras gene Proteins 0.000 description 7
- 101710163270 Nuclease Proteins 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 210000000349 chromosome Anatomy 0.000 description 7
- 239000000539 dimer Substances 0.000 description 7
- 230000006801 homologous recombination Effects 0.000 description 7
- 238000002744 homologous recombination Methods 0.000 description 7
- 230000000670 limiting effect Effects 0.000 description 7
- 230000033616 DNA repair Effects 0.000 description 6
- 108091008794 FGF receptors Proteins 0.000 description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 6
- 206010033128 Ovarian cancer Diseases 0.000 description 6
- 201000000582 Retinoblastoma Diseases 0.000 description 6
- 230000030833 cell death Effects 0.000 description 6
- 230000010261 cell growth Effects 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000007614 genetic variation Effects 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 201000005202 lung cancer Diseases 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 210000004940 nucleus Anatomy 0.000 description 6
- 108700025694 p53 Genes Proteins 0.000 description 6
- 208000008443 pancreatic carcinoma Diseases 0.000 description 6
- 230000006798 recombination Effects 0.000 description 6
- 238000005215 recombination Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- -1 type B) Proteins 0.000 description 6
- 206010069754 Acquired gene mutation Diseases 0.000 description 5
- 102100028914 Catenin beta-1 Human genes 0.000 description 5
- 108010042407 Endonucleases Proteins 0.000 description 5
- 102100030550 Menin Human genes 0.000 description 5
- 102000001760 Notch3 Receptor Human genes 0.000 description 5
- 102100033237 Pro-epidermal growth factor Human genes 0.000 description 5
- 206010060862 Prostate cancer Diseases 0.000 description 5
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 5
- 102100037282 Secretoglobin family 1D member 4 Human genes 0.000 description 5
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 5
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 5
- 101150084041 WT1 gene Proteins 0.000 description 5
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 5
- 238000009227 behaviour therapy Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 5
- 230000006780 non-homologous end joining Effects 0.000 description 5
- 201000002528 pancreatic cancer Diseases 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000037439 somatic mutation Effects 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 4
- 101100290380 Caenorhabditis elegans cel-1 gene Proteins 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 4
- 108010078791 Carrier Proteins Proteins 0.000 description 4
- 102100032616 Caspase-2 Human genes 0.000 description 4
- 102100038918 Caspase-6 Human genes 0.000 description 4
- 102100026550 Caspase-9 Human genes 0.000 description 4
- 102000011727 Caspases Human genes 0.000 description 4
- 108010076667 Caspases Proteins 0.000 description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 4
- 108010051219 Cre recombinase Proteins 0.000 description 4
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 4
- 102100036252 Cyclin-dependent kinase 4 Human genes 0.000 description 4
- 101710113436 GTPase KRas Proteins 0.000 description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 4
- 101000697486 Homo sapiens BRCA1-associated RING domain protein 1 Proteins 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 101710169972 Menin Proteins 0.000 description 4
- 108700011259 MicroRNAs Proteins 0.000 description 4
- 208000008770 Multiple Hamartoma Syndrome Diseases 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 102000001753 Notch4 Receptor Human genes 0.000 description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 description 4
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 210000004204 blood vessel Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 208000029742 colonic neoplasm Diseases 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000005782 double-strand break Effects 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000002679 microRNA Substances 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 230000005740 tumor formation Effects 0.000 description 4
- 201000010028 Acrocephalosyndactylia Diseases 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 3
- 108010014223 Armadillo Domain Proteins Proteins 0.000 description 3
- 102000002804 Ataxia Telangiectasia Mutated Proteins Human genes 0.000 description 3
- 108010004586 Ataxia Telangiectasia Mutated Proteins Proteins 0.000 description 3
- 102100028048 BRCA1-associated RING domain protein 1 Human genes 0.000 description 3
- 206010005003 Bladder cancer Diseases 0.000 description 3
- 208000010482 CADASIL Diseases 0.000 description 3
- 208000033221 Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy Diseases 0.000 description 3
- 241000699800 Cricetinae Species 0.000 description 3
- 108010025468 Cyclin-Dependent Kinase 6 Proteins 0.000 description 3
- 102100026804 Cyclin-dependent kinase 6 Human genes 0.000 description 3
- 230000004543 DNA replication Effects 0.000 description 3
- 241000289632 Dasypodidae Species 0.000 description 3
- 101150029707 ERBB2 gene Proteins 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 3
- 108010070047 Notch Receptors Proteins 0.000 description 3
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 3
- 102100034539 Peptidyl-prolyl cis-trans isomerase A Human genes 0.000 description 3
- 101100489893 Rattus norvegicus Abcg2 gene Proteins 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003098 androgen Substances 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000006471 dimerization reaction Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000009510 drug design Methods 0.000 description 3
- 206010017758 gastric cancer Diseases 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000035118 modified proteins Human genes 0.000 description 3
- 108091005573 modified proteins Proteins 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002853 nucleic acid probe Substances 0.000 description 3
- 230000002611 ovarian Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 210000002307 prostate Anatomy 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 229940126586 small molecule drug Drugs 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 201000011549 stomach cancer Diseases 0.000 description 3
- 230000002123 temporal effect Effects 0.000 description 3
- 201000005112 urinary bladder cancer Diseases 0.000 description 3
- 102100030388 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3 Human genes 0.000 description 2
- 102100036734 26S proteasome non-ATPase regulatory subunit 10 Human genes 0.000 description 2
- 102100040962 26S proteasome non-ATPase regulatory subunit 13 Human genes 0.000 description 2
- 102100036659 26S proteasome non-ATPase regulatory subunit 9 Human genes 0.000 description 2
- 102100029511 26S proteasome regulatory subunit 6B Human genes 0.000 description 2
- 102100029077 3-hydroxy-3-methylglutaryl-coenzyme A reductase Human genes 0.000 description 2
- 101710158485 3-hydroxy-3-methylglutaryl-coenzyme A reductase Proteins 0.000 description 2
- 102100039769 39S ribosomal protein L28, mitochondrial Human genes 0.000 description 2
- 102100038074 5'-AMP-activated protein kinase subunit beta-1 Human genes 0.000 description 2
- 102100027123 55 kDa erythrocyte membrane protein Human genes 0.000 description 2
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 2
- 108700001666 APC Genes Proteins 0.000 description 2
- 102100033350 ATP-dependent translocase ABCB1 Human genes 0.000 description 2
- 102100022997 Acidic leucine-rich nuclear phosphoprotein 32 family member A Human genes 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 108010044688 Activating Transcription Factor 2 Proteins 0.000 description 2
- 208000036762 Acute promyelocytic leukaemia Diseases 0.000 description 2
- 102100027236 Adenylate kinase isoenzyme 1 Human genes 0.000 description 2
- 201000011374 Alagille syndrome Diseases 0.000 description 2
- 102100027165 Alpha-2-macroglobulin receptor-associated protein Human genes 0.000 description 2
- 101710126837 Alpha-2-macroglobulin receptor-associated protein Proteins 0.000 description 2
- 102100040743 Alpha-crystallin B chain Human genes 0.000 description 2
- 206010056292 Androgen-Insensitivity Syndrome Diseases 0.000 description 2
- 102100034273 Annexin A7 Human genes 0.000 description 2
- 108010039940 Annexin A7 Proteins 0.000 description 2
- 241000272517 Anseriformes Species 0.000 description 2
- 108010062544 Apoptotic Protease-Activating Factor 1 Proteins 0.000 description 2
- 102100034524 Apoptotic protease-activating factor 1 Human genes 0.000 description 2
- 102100029361 Aromatase Human genes 0.000 description 2
- 206010003594 Ataxia telangiectasia Diseases 0.000 description 2
- 102000004000 Aurora Kinase A Human genes 0.000 description 2
- 108090000461 Aurora Kinase A Proteins 0.000 description 2
- 102100035634 B-cell linker protein Human genes 0.000 description 2
- 102100037140 BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like Human genes 0.000 description 2
- 108700040618 BRCA1 Genes Proteins 0.000 description 2
- 101150072950 BRCA1 gene Proteins 0.000 description 2
- 102100035584 BRCA2 and CDKN1A-interacting protein Human genes 0.000 description 2
- 101710159765 BRCA2 and CDKN1A-interacting protein Proteins 0.000 description 2
- 102000051485 Bcl-2 family Human genes 0.000 description 2
- 108700038897 Bcl-2 family Proteins 0.000 description 2
- 102100032305 Bcl-2 homologous antagonist/killer Human genes 0.000 description 2
- 102100021971 Bcl-2-interacting killer Human genes 0.000 description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 102100027991 Beta/gamma crystallin domain-containing protein 1 Human genes 0.000 description 2
- 101710154744 Beta/gamma crystallin domain-containing protein 1 Proteins 0.000 description 2
- 102100037674 Bis(5'-adenosyl)-triphosphatase Human genes 0.000 description 2
- 108010049931 Bone Morphogenetic Protein 2 Proteins 0.000 description 2
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 2
- 102100025401 Breast cancer type 1 susceptibility protein Human genes 0.000 description 2
- 102100022595 Broad substrate specificity ATP-binding cassette transporter ABCG2 Human genes 0.000 description 2
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 2
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 2
- 102100036846 C-C motif chemokine 21 Human genes 0.000 description 2
- 102100021936 C-C motif chemokine 27 Human genes 0.000 description 2
- 102100034798 CCAAT/enhancer-binding protein beta Human genes 0.000 description 2
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- 102100022617 COMM domain-containing protein 7 Human genes 0.000 description 2
- 102100025805 Cadherin-1 Human genes 0.000 description 2
- 102100035351 Cadherin-related family member 2 Human genes 0.000 description 2
- 101710148671 Cadherin-related family member 2 Proteins 0.000 description 2
- 102100025227 Calcium/calmodulin-dependent protein kinase type II subunit gamma Human genes 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 102100024533 Carcinoembryonic antigen-related cell adhesion molecule 1 Human genes 0.000 description 2
- 102100040751 Casein kinase II subunit alpha Human genes 0.000 description 2
- 102100040753 Casein kinase II subunit alpha' Human genes 0.000 description 2
- 102100027992 Casein kinase II subunit beta Human genes 0.000 description 2
- 102100035888 Caveolin-1 Human genes 0.000 description 2
- 108010072135 Cell Adhesion Molecule-1 Proteins 0.000 description 2
- 102100024649 Cell adhesion molecule 1 Human genes 0.000 description 2
- 102100031214 Centromere protein N Human genes 0.000 description 2
- 101710084071 Centromere protein N Proteins 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 102000006452 Class 5 Receptor-Like Protein Tyrosine Phosphatases Human genes 0.000 description 2
- 108010044213 Class 5 Receptor-Like Protein Tyrosine Phosphatases Proteins 0.000 description 2
- 102100040836 Claudin-1 Human genes 0.000 description 2
- 102000005221 Cleavage Stimulation Factor Human genes 0.000 description 2
- 108010081236 Cleavage Stimulation Factor Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 102100031162 Collagen alpha-1(XVIII) chain Human genes 0.000 description 2
- 102100033780 Collagen alpha-3(IV) chain Human genes 0.000 description 2
- 102100032778 Colorectal mutant cancer protein Human genes 0.000 description 2
- 101710190564 Colorectal mutant cancer protein Proteins 0.000 description 2
- 108010043471 Core Binding Factor Alpha 2 Subunit Proteins 0.000 description 2
- 108010079362 Core Binding Factor Alpha 3 Subunit Proteins 0.000 description 2
- 208000012609 Cowden disease Diseases 0.000 description 2
- 201000002847 Cowden syndrome Diseases 0.000 description 2
- 102100022786 Creatine kinase M-type Human genes 0.000 description 2
- 102100029376 Cryptochrome-1 Human genes 0.000 description 2
- 102100026280 Cryptochrome-2 Human genes 0.000 description 2
- 102100023033 Cyclic AMP-dependent transcription factor ATF-2 Human genes 0.000 description 2
- 102100026398 Cyclic AMP-responsive element-binding protein 3 Human genes 0.000 description 2
- 101710128029 Cyclic AMP-responsive element-binding protein 3 Proteins 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 108010060273 Cyclin A2 Proteins 0.000 description 2
- 108010058546 Cyclin D1 Proteins 0.000 description 2
- 102100025191 Cyclin-A2 Human genes 0.000 description 2
- 108010024986 Cyclin-Dependent Kinase 2 Proteins 0.000 description 2
- 102100036239 Cyclin-dependent kinase 2 Human genes 0.000 description 2
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 description 2
- 102100033233 Cyclin-dependent kinase inhibitor 1B Human genes 0.000 description 2
- 102100033269 Cyclin-dependent kinase inhibitor 1C Human genes 0.000 description 2
- 102100030497 Cytochrome c Human genes 0.000 description 2
- 101710103962 Cytochrome c, somatic Proteins 0.000 description 2
- 102100025269 DENN domain-containing protein 2B Human genes 0.000 description 2
- 101710141585 DENN domain-containing protein 2B Proteins 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- 102100039524 DNA endonuclease RBBP8 Human genes 0.000 description 2
- 108050008316 DNA endonuclease RBBP8 Proteins 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- 102100037799 DNA-binding protein Ikaros Human genes 0.000 description 2
- 108010031042 Death-Associated Protein Kinases Proteins 0.000 description 2
- 102100039694 Death-associated protein 1 Human genes 0.000 description 2
- 102100038587 Death-associated protein kinase 1 Human genes 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 description 2
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 2
- 102100028572 Disabled homolog 2 Human genes 0.000 description 2
- 108010038530 Dual Specificity Phosphatase 6 Proteins 0.000 description 2
- 102100028944 Dual specificity protein phosphatase 13 isoform B Human genes 0.000 description 2
- 102100027274 Dual specificity protein phosphatase 6 Human genes 0.000 description 2
- 102100021179 Dynamin-3 Human genes 0.000 description 2
- 108010063774 E2F1 Transcription Factor Proteins 0.000 description 2
- 102000005718 E2F6 Transcription Factor Human genes 0.000 description 2
- 108010031068 E2F6 Transcription Factor Proteins 0.000 description 2
- 102100032353 E3 ubiquitin-protein ligase CHFR Human genes 0.000 description 2
- 102100031918 E3 ubiquitin-protein ligase NEDD4 Human genes 0.000 description 2
- 102100021765 E3 ubiquitin-protein ligase RNF139 Human genes 0.000 description 2
- 102100021838 E3 ubiquitin-protein ligase SIAH1 Human genes 0.000 description 2
- 102100031748 E3 ubiquitin-protein ligase SIAH2 Human genes 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102100021658 Embigin Human genes 0.000 description 2
- 102100037241 Endoglin Human genes 0.000 description 2
- 108010036395 Endoglin Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 102100030322 Ephrin type-A receptor 1 Human genes 0.000 description 2
- 102100031968 Ephrin type-B receptor 2 Human genes 0.000 description 2
- 102100023733 Ephrin-B3 Human genes 0.000 description 2
- 108010044085 Ephrin-B3 Proteins 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 102100035549 Eukaryotic translation initiation factor 2 subunit 1 Human genes 0.000 description 2
- 101710196290 Eukaryotic translation initiation factor 2-alpha kinase 2 Proteins 0.000 description 2
- 102100034295 Eukaryotic translation initiation factor 3 subunit A Human genes 0.000 description 2
- 102100039735 Eukaryotic translation initiation factor 4 gamma 1 Human genes 0.000 description 2
- 102100039737 Eukaryotic translation initiation factor 4 gamma 2 Human genes 0.000 description 2
- 102000008968 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 description 2
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 2
- 102100040002 Eukaryotic translation initiation factor 6 Human genes 0.000 description 2
- 101710204615 Eukaryotic translation initiation factor 6 Proteins 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 102100028138 F-box/WD repeat-containing protein 7 Human genes 0.000 description 2
- 201000001342 Fallopian tube cancer Diseases 0.000 description 2
- 208000013452 Fallopian tube neoplasm Diseases 0.000 description 2
- 201000006107 Familial adenomatous polyposis Diseases 0.000 description 2
- 208000015103 Familial isolated hyperparathyroidism Diseases 0.000 description 2
- 102000016627 Fanconi Anemia Complementation Group N protein Human genes 0.000 description 2
- 108010067741 Fanconi Anemia Complementation Group N protein Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 description 2
- 101710182386 Fibroblast growth factor receptor 1 Proteins 0.000 description 2
- 102100037362 Fibronectin Human genes 0.000 description 2
- 102100028314 Filaggrin Human genes 0.000 description 2
- 101710088660 Filaggrin Proteins 0.000 description 2
- 102000016970 Follistatin Human genes 0.000 description 2
- 108010014612 Follistatin Proteins 0.000 description 2
- 102100035427 Forkhead box protein O1 Human genes 0.000 description 2
- 102100035421 Forkhead box protein O3 Human genes 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 102100024165 G1/S-specific cyclin-D1 Human genes 0.000 description 2
- 102100039558 Galectin-3 Human genes 0.000 description 2
- 102100021337 Gap junction alpha-1 protein Human genes 0.000 description 2
- 102100037260 Gap junction beta-1 protein Human genes 0.000 description 2
- 102000004878 Gelsolin Human genes 0.000 description 2
- 108090001064 Gelsolin Proteins 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 102100022630 Glutamate receptor ionotropic, NMDA 2B Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 2
- 102100038104 Glycogen synthase kinase-3 beta Human genes 0.000 description 2
- 102100031154 Growth arrest and DNA damage-inducible protein GADD45 gamma Human genes 0.000 description 2
- 102100036683 Growth arrest-specific protein 1 Human genes 0.000 description 2
- 206010053759 Growth retardation Diseases 0.000 description 2
- 102100039330 HMG box-containing protein 1 Human genes 0.000 description 2
- 101710201214 HMG box-containing protein 1 Proteins 0.000 description 2
- 102100031561 Hamartin Human genes 0.000 description 2
- 101710175981 Hamartin Proteins 0.000 description 2
- 102100028765 Heat shock 70 kDa protein 4 Human genes 0.000 description 2
- 102100027421 Heat shock cognate 71 kDa protein Human genes 0.000 description 2
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 2
- 102100039165 Heat shock protein beta-1 Human genes 0.000 description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 2
- 108010024124 Histone Deacetylase 1 Proteins 0.000 description 2
- 102100034533 Histone H2AX Human genes 0.000 description 2
- 102100022846 Histone acetyltransferase KAT2B Human genes 0.000 description 2
- 102100039996 Histone deacetylase 1 Human genes 0.000 description 2
- 102100021454 Histone deacetylase 4 Human genes 0.000 description 2
- 101710177324 Histone deacetylase 4 Proteins 0.000 description 2
- 102100038720 Histone deacetylase 9 Human genes 0.000 description 2
- 101710177326 Histone deacetylase 9 Proteins 0.000 description 2
- 102100038970 Histone-lysine N-methyltransferase EZH2 Human genes 0.000 description 2
- 102100028092 Homeobox protein Nkx-3.1 Human genes 0.000 description 2
- 102100035081 Homeobox protein TGIF1 Human genes 0.000 description 2
- 102100028798 Homeodomain-only protein Human genes 0.000 description 2
- 101000583069 Homo sapiens 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3 Proteins 0.000 description 2
- 101001136581 Homo sapiens 26S proteasome non-ATPase regulatory subunit 10 Proteins 0.000 description 2
- 101000612536 Homo sapiens 26S proteasome non-ATPase regulatory subunit 13 Proteins 0.000 description 2
- 101001136710 Homo sapiens 26S proteasome non-ATPase regulatory subunit 9 Proteins 0.000 description 2
- 101001125524 Homo sapiens 26S proteasome regulatory subunit 6B Proteins 0.000 description 2
- 101000667524 Homo sapiens 39S ribosomal protein L28, mitochondrial Proteins 0.000 description 2
- 101000742701 Homo sapiens 5'-AMP-activated protein kinase subunit beta-1 Proteins 0.000 description 2
- 101001057956 Homo sapiens 55 kDa erythrocyte membrane protein Proteins 0.000 description 2
- 101000883686 Homo sapiens 60 kDa heat shock protein, mitochondrial Proteins 0.000 description 2
- 101000757200 Homo sapiens Acidic leucine-rich nuclear phosphoprotein 32 family member A Proteins 0.000 description 2
- 101000891982 Homo sapiens Alpha-crystallin B chain Proteins 0.000 description 2
- 101000919395 Homo sapiens Aromatase Proteins 0.000 description 2
- 101000803266 Homo sapiens B-cell linker protein Proteins 0.000 description 2
- 101000740545 Homo sapiens BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like Proteins 0.000 description 2
- 101000798320 Homo sapiens Bcl-2 homologous antagonist/killer Proteins 0.000 description 2
- 101000970576 Homo sapiens Bcl-2-interacting killer Proteins 0.000 description 2
- 101000765010 Homo sapiens Beta-galactosidase Proteins 0.000 description 2
- 101001027506 Homo sapiens Bis(5'-adenosyl)-triphosphatase Proteins 0.000 description 2
- 101000934870 Homo sapiens Breast cancer type 1 susceptibility protein Proteins 0.000 description 2
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 2
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 2
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 description 2
- 101000897494 Homo sapiens C-C motif chemokine 27 Proteins 0.000 description 2
- 101000945963 Homo sapiens CCAAT/enhancer-binding protein beta Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 2
- 101000899983 Homo sapiens COMM domain-containing protein 7 Proteins 0.000 description 2
- 101001077334 Homo sapiens Calcium/calmodulin-dependent protein kinase type II subunit gamma Proteins 0.000 description 2
- 101000892026 Homo sapiens Casein kinase II subunit alpha Proteins 0.000 description 2
- 101000892015 Homo sapiens Casein kinase II subunit alpha' Proteins 0.000 description 2
- 101000858625 Homo sapiens Casein kinase II subunit beta Proteins 0.000 description 2
- 101000983528 Homo sapiens Caspase-8 Proteins 0.000 description 2
- 101000916173 Homo sapiens Catenin beta-1 Proteins 0.000 description 2
- 101000715467 Homo sapiens Caveolin-1 Proteins 0.000 description 2
- 101000940068 Homo sapiens Collagen alpha-1(XVIII) chain Proteins 0.000 description 2
- 101000710873 Homo sapiens Collagen alpha-3(IV) chain Proteins 0.000 description 2
- 101000919351 Homo sapiens Cryptochrome-1 Proteins 0.000 description 2
- 101000855613 Homo sapiens Cryptochrome-2 Proteins 0.000 description 2
- 101000599038 Homo sapiens DNA-binding protein Ikaros Proteins 0.000 description 2
- 101000886250 Homo sapiens Death-associated protein 1 Proteins 0.000 description 2
- 101000915391 Homo sapiens Disabled homolog 2 Proteins 0.000 description 2
- 101000838551 Homo sapiens Dual specificity protein phosphatase 13 isoform A Proteins 0.000 description 2
- 101000838549 Homo sapiens Dual specificity protein phosphatase 13 isoform B Proteins 0.000 description 2
- 101000942970 Homo sapiens E3 ubiquitin-protein ligase CHFR Proteins 0.000 description 2
- 101000636713 Homo sapiens E3 ubiquitin-protein ligase NEDD4 Proteins 0.000 description 2
- 101000616722 Homo sapiens E3 ubiquitin-protein ligase SIAH1 Proteins 0.000 description 2
- 101000707245 Homo sapiens E3 ubiquitin-protein ligase SIAH2 Proteins 0.000 description 2
- 101000896275 Homo sapiens Embigin Proteins 0.000 description 2
- 101000938354 Homo sapiens Ephrin type-A receptor 1 Proteins 0.000 description 2
- 101001064462 Homo sapiens Ephrin type-B receptor 2 Proteins 0.000 description 2
- 101001020112 Homo sapiens Eukaryotic translation initiation factor 2 subunit 1 Proteins 0.000 description 2
- 101000925957 Homo sapiens Eukaryotic translation initiation factor 3 subunit A Proteins 0.000 description 2
- 101001034825 Homo sapiens Eukaryotic translation initiation factor 4 gamma 1 Proteins 0.000 description 2
- 101001034811 Homo sapiens Eukaryotic translation initiation factor 4 gamma 2 Proteins 0.000 description 2
- 101001060231 Homo sapiens F-box/WD repeat-containing protein 7 Proteins 0.000 description 2
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 2
- 101000877727 Homo sapiens Forkhead box protein O1 Proteins 0.000 description 2
- 101000877681 Homo sapiens Forkhead box protein O3 Proteins 0.000 description 2
- 101000930963 Homo sapiens Forkhead box protein O3B Proteins 0.000 description 2
- 101000608757 Homo sapiens Galectin-3 Proteins 0.000 description 2
- 101000894966 Homo sapiens Gap junction alpha-1 protein Proteins 0.000 description 2
- 101000954104 Homo sapiens Gap junction beta-1 protein Proteins 0.000 description 2
- 101001066163 Homo sapiens Growth arrest and DNA damage-inducible protein GADD45 gamma Proteins 0.000 description 2
- 101001072723 Homo sapiens Growth arrest-specific protein 1 Proteins 0.000 description 2
- 101001078692 Homo sapiens Heat shock 70 kDa protein 4 Proteins 0.000 description 2
- 101001080568 Homo sapiens Heat shock cognate 71 kDa protein Proteins 0.000 description 2
- 101001016865 Homo sapiens Heat shock protein HSP 90-alpha Proteins 0.000 description 2
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 description 2
- 101001067891 Homo sapiens Histone H2AX Proteins 0.000 description 2
- 101001047006 Homo sapiens Histone acetyltransferase KAT2B Proteins 0.000 description 2
- 101000882127 Homo sapiens Histone-lysine N-methyltransferase EZH2 Proteins 0.000 description 2
- 101000578249 Homo sapiens Homeobox protein Nkx-3.1 Proteins 0.000 description 2
- 101000596925 Homo sapiens Homeobox protein TGIF1 Proteins 0.000 description 2
- 101000839095 Homo sapiens Homeodomain-only protein Proteins 0.000 description 2
- 101000983515 Homo sapiens Inactive caspase-12 Proteins 0.000 description 2
- 101001056180 Homo sapiens Induced myeloid leukemia cell differentiation protein Mcl-1 Proteins 0.000 description 2
- 101001054334 Homo sapiens Interferon beta Proteins 0.000 description 2
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 description 2
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 2
- 101000971521 Homo sapiens Kinetochore scaffold 1 Proteins 0.000 description 2
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 2
- 101001004821 Homo sapiens Late cornified envelope-like proline-rich protein 1 Proteins 0.000 description 2
- 101001088892 Homo sapiens Lysine-specific demethylase 5A Proteins 0.000 description 2
- 101001088883 Homo sapiens Lysine-specific demethylase 5B Proteins 0.000 description 2
- 101001043321 Homo sapiens Lysyl oxidase homolog 1 Proteins 0.000 description 2
- 101001043352 Homo sapiens Lysyl oxidase homolog 2 Proteins 0.000 description 2
- 101001043354 Homo sapiens Lysyl oxidase homolog 3 Proteins 0.000 description 2
- 101001043351 Homo sapiens Lysyl oxidase homolog 4 Proteins 0.000 description 2
- 101001056128 Homo sapiens Mannose-binding protein C Proteins 0.000 description 2
- 101000954986 Homo sapiens Merlin Proteins 0.000 description 2
- 101000645293 Homo sapiens Metalloproteinase inhibitor 3 Proteins 0.000 description 2
- 101000794228 Homo sapiens Mitotic checkpoint serine/threonine-protein kinase BUB1 beta Proteins 0.000 description 2
- 101000957106 Homo sapiens Mitotic spindle assembly checkpoint protein MAD1 Proteins 0.000 description 2
- 101000972284 Homo sapiens Mucin-3A Proteins 0.000 description 2
- 101000906927 Homo sapiens N-chimaerin Proteins 0.000 description 2
- 101000636665 Homo sapiens NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 Proteins 0.000 description 2
- 101000961071 Homo sapiens NF-kappa-B inhibitor alpha Proteins 0.000 description 2
- 101001123834 Homo sapiens Neprilysin Proteins 0.000 description 2
- 101000978766 Homo sapiens Neurogenic locus notch homolog protein 1 Proteins 0.000 description 2
- 101001124991 Homo sapiens Nitric oxide synthase, inducible Proteins 0.000 description 2
- 101001109719 Homo sapiens Nucleophosmin Proteins 0.000 description 2
- 101000839399 Homo sapiens Oxidoreductase HTATIP2 Proteins 0.000 description 2
- 101001123306 Homo sapiens PR domain zinc finger protein 10 Proteins 0.000 description 2
- 101000601661 Homo sapiens Paired box protein Pax-7 Proteins 0.000 description 2
- 101000945735 Homo sapiens Parafibromin Proteins 0.000 description 2
- 101000693243 Homo sapiens Paternally-expressed gene 3 protein Proteins 0.000 description 2
- 101001073216 Homo sapiens Period circadian protein homolog 2 Proteins 0.000 description 2
- 101000692259 Homo sapiens Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 Proteins 0.000 description 2
- 101000609261 Homo sapiens Plasminogen activator inhibitor 2 Proteins 0.000 description 2
- 101000583714 Homo sapiens Pleckstrin homology-like domain family A member 3 Proteins 0.000 description 2
- 101000613343 Homo sapiens Polycomb group RING finger protein 2 Proteins 0.000 description 2
- 101000613355 Homo sapiens Polycomb group RING finger protein 6 Proteins 0.000 description 2
- 101001074444 Homo sapiens Polycystin-1 Proteins 0.000 description 2
- 101000945496 Homo sapiens Proliferation marker protein Ki-67 Proteins 0.000 description 2
- 101000577696 Homo sapiens Proline-rich transmembrane protein 2 Proteins 0.000 description 2
- 101001046603 Homo sapiens Protein KIBRA Proteins 0.000 description 2
- 101000772227 Homo sapiens Protein TSSC4 Proteins 0.000 description 2
- 101000804792 Homo sapiens Protein Wnt-5a Proteins 0.000 description 2
- 101000861454 Homo sapiens Protein c-Fos Proteins 0.000 description 2
- 101001051777 Homo sapiens Protein kinase C alpha type Proteins 0.000 description 2
- 101001051767 Homo sapiens Protein kinase C beta type Proteins 0.000 description 2
- 101001026864 Homo sapiens Protein kinase C gamma type Proteins 0.000 description 2
- 101001098560 Homo sapiens Proteinase-activated receptor 2 Proteins 0.000 description 2
- 101000655540 Homo sapiens Protransforming growth factor alpha Proteins 0.000 description 2
- 101001132698 Homo sapiens Retinoic acid receptor beta Proteins 0.000 description 2
- 101001106322 Homo sapiens Rho GTPase-activating protein 7 Proteins 0.000 description 2
- 101000581112 Homo sapiens Rho-related GTP-binding protein RhoB Proteins 0.000 description 2
- 101000708835 Homo sapiens SET and MYND domain-containing protein 4 Proteins 0.000 description 2
- 101000880431 Homo sapiens Serine/threonine-protein kinase 4 Proteins 0.000 description 2
- 101001047642 Homo sapiens Serine/threonine-protein kinase LATS1 Proteins 0.000 description 2
- 101000576901 Homo sapiens Serine/threonine-protein kinase MRCK alpha Proteins 0.000 description 2
- 101000628562 Homo sapiens Serine/threonine-protein kinase STK11 Proteins 0.000 description 2
- 101000651890 Homo sapiens Slit homolog 2 protein Proteins 0.000 description 2
- 101001131204 Homo sapiens Sulfhydryl oxidase 1 Proteins 0.000 description 2
- 101000799181 Homo sapiens TP53-binding protein 1 Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 101000702545 Homo sapiens Transcription activator BRG1 Proteins 0.000 description 2
- 101001051166 Homo sapiens Transcriptional activator MN1 Proteins 0.000 description 2
- 101000635938 Homo sapiens Transforming growth factor beta-1 proprotein Proteins 0.000 description 2
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 2
- 101000830570 Homo sapiens Tumor necrosis factor alpha-induced protein 3 Proteins 0.000 description 2
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 description 2
- 101000772122 Homo sapiens Twisted gastrulation protein homolog 1 Proteins 0.000 description 2
- 101000864342 Homo sapiens Tyrosine-protein kinase BTK Proteins 0.000 description 2
- 101000671649 Homo sapiens Upstream stimulatory factor 2 Proteins 0.000 description 2
- 101000638886 Homo sapiens Urokinase-type plasminogen activator Proteins 0.000 description 2
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 description 2
- 101000621390 Homo sapiens Wee1-like protein kinase Proteins 0.000 description 2
- 101000964562 Homo sapiens Zinc finger FYVE domain-containing protein 9 Proteins 0.000 description 2
- 101000795753 Homo sapiens mRNA decay activator protein ZFP36 Proteins 0.000 description 2
- 102100039283 Hyaluronidase-1 Human genes 0.000 description 2
- 102100039285 Hyaluronidase-2 Human genes 0.000 description 2
- 102100021082 Hyaluronidase-3 Human genes 0.000 description 2
- 102100026556 Inactive caspase-12 Human genes 0.000 description 2
- 102100026539 Induced myeloid leukemia cell differentiation protein Mcl-1 Human genes 0.000 description 2
- 102100036721 Insulin receptor Human genes 0.000 description 2
- 102100029228 Insulin-like growth factor-binding protein 7 Human genes 0.000 description 2
- 102000012334 Integrin beta4 Human genes 0.000 description 2
- 108010022238 Integrin beta4 Proteins 0.000 description 2
- 102100020944 Integrin-linked protein kinase Human genes 0.000 description 2
- 102100026720 Interferon beta Human genes 0.000 description 2
- 102100036981 Interferon regulatory factor 1 Human genes 0.000 description 2
- 108090000890 Interferon regulatory factor 1 Proteins 0.000 description 2
- 102100034170 Interferon-induced, double-stranded RNA-activated protein kinase Human genes 0.000 description 2
- 101710089751 Interferon-induced, double-stranded RNA-activated protein kinase Proteins 0.000 description 2
- 102100036671 Interleukin-24 Human genes 0.000 description 2
- 102100039064 Interleukin-3 Human genes 0.000 description 2
- 102100033501 Interleukin-32 Human genes 0.000 description 2
- 102000012322 Junction plakoglobin Human genes 0.000 description 2
- 102100040445 Keratin, type I cytoskeletal 14 Human genes 0.000 description 2
- 102100022905 Keratin, type II cytoskeletal 1 Human genes 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- 102100021464 Kinetochore scaffold 1 Human genes 0.000 description 2
- 102100020880 Kit ligand Human genes 0.000 description 2
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 102100025972 Late cornified envelope-like proline-rich protein 1 Human genes 0.000 description 2
- 201000011062 Li-Fraumeni syndrome Diseases 0.000 description 2
- 102100033246 Lysine-specific demethylase 5A Human genes 0.000 description 2
- 102100033247 Lysine-specific demethylase 5B Human genes 0.000 description 2
- 108010009491 Lysosomal-Associated Membrane Protein 2 Proteins 0.000 description 2
- 102100038225 Lysosome-associated membrane glycoprotein 2 Human genes 0.000 description 2
- 102100021958 Lysyl oxidase homolog 1 Human genes 0.000 description 2
- 102100021948 Lysyl oxidase homolog 2 Human genes 0.000 description 2
- 102100021949 Lysyl oxidase homolog 3 Human genes 0.000 description 2
- 102100021968 Lysyl oxidase homolog 4 Human genes 0.000 description 2
- 102100028396 MAP kinase-activated protein kinase 5 Human genes 0.000 description 2
- 101710140998 MAP kinase-activated protein kinase 5 Proteins 0.000 description 2
- 108010059343 MM Form Creatine Kinase Proteins 0.000 description 2
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 2
- 208000004059 Male Breast Neoplasms Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 102100026553 Mannose-binding protein C Human genes 0.000 description 2
- 208000036626 Mental retardation Diseases 0.000 description 2
- 102100037106 Merlin Human genes 0.000 description 2
- 102100026261 Metalloproteinase inhibitor 3 Human genes 0.000 description 2
- 108700027654 Mitogen-Activated Protein Kinase 10 Proteins 0.000 description 2
- 108700036166 Mitogen-Activated Protein Kinase 11 Proteins 0.000 description 2
- 108700027648 Mitogen-Activated Protein Kinase 8 Proteins 0.000 description 2
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 2
- 102100026931 Mitogen-activated protein kinase 10 Human genes 0.000 description 2
- 102100026929 Mitogen-activated protein kinase 11 Human genes 0.000 description 2
- 108700015928 Mitogen-activated protein kinase 13 Proteins 0.000 description 2
- 102100037808 Mitogen-activated protein kinase 8 Human genes 0.000 description 2
- 102100030144 Mitotic checkpoint serine/threonine-protein kinase BUB1 beta Human genes 0.000 description 2
- 102100038828 Mitotic spindle assembly checkpoint protein MAD1 Human genes 0.000 description 2
- 102000010909 Monoamine Oxidase Human genes 0.000 description 2
- 108010062431 Monoamine oxidase Proteins 0.000 description 2
- 102100025748 Mothers against decapentaplegic homolog 3 Human genes 0.000 description 2
- 101710143111 Mothers against decapentaplegic homolog 3 Proteins 0.000 description 2
- 102100025725 Mothers against decapentaplegic homolog 4 Human genes 0.000 description 2
- 101710143112 Mothers against decapentaplegic homolog 4 Proteins 0.000 description 2
- 102100022497 Mucin-3A Human genes 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 206010062575 Muscle contracture Diseases 0.000 description 2
- 102100024134 Myeloid differentiation primary response protein MyD88 Human genes 0.000 description 2
- 108010081823 Myocardin Proteins 0.000 description 2
- 102100030217 Myocardin Human genes 0.000 description 2
- 108010052185 Myotonin-Protein Kinase Proteins 0.000 description 2
- 102100022437 Myotonin-protein kinase Human genes 0.000 description 2
- 102100023648 N-chimaerin Human genes 0.000 description 2
- 102100031924 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 Human genes 0.000 description 2
- 102100039337 NF-kappa-B inhibitor alpha Human genes 0.000 description 2
- 108010054200 NR2B NMDA receptor Proteins 0.000 description 2
- 102100028782 Neprilysin Human genes 0.000 description 2
- 102000009065 Netrin-1 Human genes 0.000 description 2
- 108010074223 Netrin-1 Proteins 0.000 description 2
- 102100024403 Nibrin Human genes 0.000 description 2
- 108050003990 Nibrin Proteins 0.000 description 2
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 2
- 102000005650 Notch Receptors Human genes 0.000 description 2
- 108010029755 Notch1 Receptor Proteins 0.000 description 2
- 102000001756 Notch2 Receptor Human genes 0.000 description 2
- 108010029751 Notch2 Receptor Proteins 0.000 description 2
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 2
- 102100023050 Nuclear factor NF-kappa-B p105 subunit Human genes 0.000 description 2
- 101710082694 Nuclear factor NF-kappa-B p105 subunit Proteins 0.000 description 2
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 2
- 102100022678 Nucleophosmin Human genes 0.000 description 2
- 108010053291 Oncogene Protein v-akt Proteins 0.000 description 2
- 102100021079 Ornithine decarboxylase Human genes 0.000 description 2
- 101710120430 Ornithine decarboxylase 1 Proteins 0.000 description 2
- 102100027952 Oxidoreductase HTATIP2 Human genes 0.000 description 2
- 102100028955 PR domain zinc finger protein 10 Human genes 0.000 description 2
- 201000010917 PTEN hamartoma tumor syndrome Diseases 0.000 description 2
- 102100037504 Paired box protein Pax-5 Human genes 0.000 description 2
- 102100037503 Paired box protein Pax-7 Human genes 0.000 description 2
- 102100034743 Parafibromin Human genes 0.000 description 2
- 102100025757 Paternally-expressed gene 3 protein Human genes 0.000 description 2
- 102000018546 Paxillin Human genes 0.000 description 2
- ACNHBCIZLNNLRS-UHFFFAOYSA-N Paxilline 1 Natural products N1C2=CC=CC=C2C2=C1C1(C)C3(C)CCC4OC(C(C)(O)C)C(=O)C=C4C3(O)CCC1C2 ACNHBCIZLNNLRS-UHFFFAOYSA-N 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102100035787 Period circadian protein homolog 2 Human genes 0.000 description 2
- 201000004014 Pfeiffer syndrome Diseases 0.000 description 2
- 102100026066 Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 Human genes 0.000 description 2
- 102100039419 Plasminogen activator inhibitor 2 Human genes 0.000 description 2
- 102100030925 Pleckstrin homology-like domain family A member 3 Human genes 0.000 description 2
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 2
- 102100040919 Polycomb group RING finger protein 2 Human genes 0.000 description 2
- 102100040917 Polycomb group RING finger protein 6 Human genes 0.000 description 2
- 102100036143 Polycystin-1 Human genes 0.000 description 2
- 102100037935 Polyubiquitin-C Human genes 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 108010069820 Pro-Opiomelanocortin Proteins 0.000 description 2
- 239000000683 Pro-Opiomelanocortin Substances 0.000 description 2
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 2
- 102100026534 Procathepsin L Human genes 0.000 description 2
- 102100025803 Progesterone receptor Human genes 0.000 description 2
- 102100040126 Prokineticin-1 Human genes 0.000 description 2
- 102100036691 Proliferating cell nuclear antigen Human genes 0.000 description 2
- 102100034836 Proliferation marker protein Ki-67 Human genes 0.000 description 2
- 102100028840 Proline-rich transmembrane protein 2 Human genes 0.000 description 2
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100022309 Protein KIBRA Human genes 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 102100023089 Protein S100-A2 Human genes 0.000 description 2
- 102000009516 Protein Serine-Threonine Kinases Human genes 0.000 description 2
- 108010009341 Protein Serine-Threonine Kinases Proteins 0.000 description 2
- 102100029345 Protein TSSC4 Human genes 0.000 description 2
- 102100027584 Protein c-Fos Human genes 0.000 description 2
- 102100024924 Protein kinase C alpha type Human genes 0.000 description 2
- 102100024923 Protein kinase C beta type Human genes 0.000 description 2
- 102100037314 Protein kinase C gamma type Human genes 0.000 description 2
- 102000017332 Protein kinase C, delta Human genes 0.000 description 2
- 108050005326 Protein kinase C, delta Proteins 0.000 description 2
- 102000015840 Protein kinase C, epsilon Human genes 0.000 description 2
- 108050004067 Protein kinase C, epsilon Proteins 0.000 description 2
- 108010003894 Protein-Lysine 6-Oxidase Proteins 0.000 description 2
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 2
- 102100026858 Protein-lysine 6-oxidase Human genes 0.000 description 2
- 102100037132 Proteinase-activated receptor 2 Human genes 0.000 description 2
- 208000007531 Proteus syndrome Diseases 0.000 description 2
- 102000008022 Proto-Oncogene Proteins c-met Human genes 0.000 description 2
- 108010089836 Proto-Oncogene Proteins c-met Proteins 0.000 description 2
- 102100032350 Protransforming growth factor alpha Human genes 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 108010068097 Rad51 Recombinase Proteins 0.000 description 2
- 102000002490 Rad51 Recombinase Human genes 0.000 description 2
- 101150002130 Rb1 gene Proteins 0.000 description 2
- 102100029771 Remodeling and spacing factor 1 Human genes 0.000 description 2
- 101710201736 Remodeling and spacing factor 1 Proteins 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 102100029831 Reticulon-4 Human genes 0.000 description 2
- 102100033909 Retinoic acid receptor beta Human genes 0.000 description 2
- 102100021433 Rho GTPase-activating protein 1 Human genes 0.000 description 2
- 101710116876 Rho GTPase-activating protein 1 Proteins 0.000 description 2
- 102100021446 Rho GTPase-activating protein 7 Human genes 0.000 description 2
- 102100039313 Rho-associated protein kinase 1 Human genes 0.000 description 2
- 101710088411 Rho-associated protein kinase 1 Proteins 0.000 description 2
- 102100039314 Rho-associated protein kinase 2 Human genes 0.000 description 2
- 101710088493 Rho-associated protein kinase 2 Proteins 0.000 description 2
- 102100027611 Rho-related GTP-binding protein RhoB Human genes 0.000 description 2
- 102100025373 Runt-related transcription factor 1 Human genes 0.000 description 2
- 102100025369 Runt-related transcription factor 3 Human genes 0.000 description 2
- 102100034374 S-phase kinase-associated protein 2 Human genes 0.000 description 2
- 102100032725 SET and MYND domain-containing protein 4 Human genes 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 102100037629 Serine/threonine-protein kinase 4 Human genes 0.000 description 2
- 102100024031 Serine/threonine-protein kinase LATS1 Human genes 0.000 description 2
- 102100025352 Serine/threonine-protein kinase MRCK alpha Human genes 0.000 description 2
- 102100032771 Serine/threonine-protein kinase SIK1 Human genes 0.000 description 2
- 101710083834 Serine/threonine-protein kinase SIK1 Proteins 0.000 description 2
- 102100026715 Serine/threonine-protein kinase STK11 Human genes 0.000 description 2
- 102100023085 Serine/threonine-protein kinase mTOR Human genes 0.000 description 2
- 102100030333 Serpin B5 Human genes 0.000 description 2
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 2
- 102100027340 Slit homolog 2 protein Human genes 0.000 description 2
- 102100032799 Smoothened homolog Human genes 0.000 description 2
- 102100027215 Sodium-coupled monocarboxylate transporter 1 Human genes 0.000 description 2
- 102100023102 Solute carrier family 22 member 18 Human genes 0.000 description 2
- 102100034371 Sulfhydryl oxidase 1 Human genes 0.000 description 2
- 102100038836 Superoxide dismutase [Cu-Zn] Human genes 0.000 description 2
- 101710139715 Superoxide dismutase [Cu-Zn] Proteins 0.000 description 2
- 102100032891 Superoxide dismutase [Mn], mitochondrial Human genes 0.000 description 2
- 101710202572 Superoxide dismutase [Mn], mitochondrial Proteins 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 108010016672 Syk Kinase Proteins 0.000 description 2
- 102100031115 Syntaxin-11 Human genes 0.000 description 2
- 208000029052 T-cell acute lymphoblastic leukemia Diseases 0.000 description 2
- 102100033455 TGF-beta receptor type-2 Human genes 0.000 description 2
- 102100034107 TP53-binding protein 1 Human genes 0.000 description 2
- 108010017842 Telomerase Proteins 0.000 description 2
- 102100032938 Telomerase reverse transcriptase Human genes 0.000 description 2
- 102100024547 Tensin-1 Human genes 0.000 description 2
- 102100024545 Tensin-4 Human genes 0.000 description 2
- 102100034915 Tetraspanin-32 Human genes 0.000 description 2
- 108700031954 Tgfb1i1/Leupaxin/TGFB1I1 Proteins 0.000 description 2
- 102100031344 Thioredoxin-interacting protein Human genes 0.000 description 2
- 101710114149 Thioredoxin-interacting protein Proteins 0.000 description 2
- 102000002938 Thrombospondin Human genes 0.000 description 2
- 108060008245 Thrombospondin Proteins 0.000 description 2
- 102100036034 Thrombospondin-1 Human genes 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- 108010048992 Transcription Factor 4 Proteins 0.000 description 2
- 102100031027 Transcription activator BRG1 Human genes 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102100023489 Transcription factor 4 Human genes 0.000 description 2
- 102100024026 Transcription factor E2F1 Human genes 0.000 description 2
- 102100024592 Transcriptional activator MN1 Human genes 0.000 description 2
- 102100031873 Transcriptional coactivator YAP1 Human genes 0.000 description 2
- 101710193680 Transcriptional coactivator YAP1 Proteins 0.000 description 2
- 108010011702 Transforming Growth Factor-beta Type I Receptor Proteins 0.000 description 2
- 102000014172 Transforming Growth Factor-beta Type I Receptor Human genes 0.000 description 2
- 102100030742 Transforming growth factor beta-1 proprotein Human genes 0.000 description 2
- 102100031638 Tuberin Human genes 0.000 description 2
- 108050009309 Tuberin Proteins 0.000 description 2
- 102000018252 Tumor Protein p73 Human genes 0.000 description 2
- 108010091356 Tumor Protein p73 Proteins 0.000 description 2
- 102100040247 Tumor necrosis factor Human genes 0.000 description 2
- 102100024596 Tumor necrosis factor alpha-induced protein 3 Human genes 0.000 description 2
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 2
- 102100029320 Twisted gastrulation protein homolog 1 Human genes 0.000 description 2
- 102100039094 Tyrosinase Human genes 0.000 description 2
- 102100029823 Tyrosine-protein kinase BTK Human genes 0.000 description 2
- 102100038183 Tyrosine-protein kinase SYK Human genes 0.000 description 2
- 108010056354 Ubiquitin C Proteins 0.000 description 2
- 102100021013 Ubiquitin carboxyl-terminal hydrolase 7 Human genes 0.000 description 2
- 102100040103 Upstream stimulatory factor 2 Human genes 0.000 description 2
- 108010042352 Urokinase Plasminogen Activator Receptors Proteins 0.000 description 2
- 102100024689 Urokinase plasminogen activator surface receptor Human genes 0.000 description 2
- 102100031358 Urokinase-type plasminogen activator Human genes 0.000 description 2
- 102000003848 Uteroglobin Human genes 0.000 description 2
- 108090000203 Uteroglobin Proteins 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000009520 Vascular Endothelial Growth Factor C Human genes 0.000 description 2
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 2
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 2
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 2
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 2
- 102100023037 Wee1-like protein kinase Human genes 0.000 description 2
- 208000008383 Wilms tumor Diseases 0.000 description 2
- 102000043366 Wnt-5a Human genes 0.000 description 2
- 102100040801 Zinc finger FYVE domain-containing protein 9 Human genes 0.000 description 2
- 102100023406 Zinc finger RNA-binding protein Human genes 0.000 description 2
- 101710150392 Zinc finger RNA-binding protein Proteins 0.000 description 2
- 101710185494 Zinc finger protein Proteins 0.000 description 2
- 102100023597 Zinc finger protein 816 Human genes 0.000 description 2
- 102100035558 Zinc finger protein GLI2 Human genes 0.000 description 2
- 208000008919 achondroplasia Diseases 0.000 description 2
- 108010066695 adenylate kinase 1 Proteins 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- 101150024147 bax gene Proteins 0.000 description 2
- 102000000472 beta-Transducin Repeat-Containing Proteins Human genes 0.000 description 2
- 108010080842 beta-Transducin Repeat-Containing Proteins Proteins 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 208000006111 contracture Diseases 0.000 description 2
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108010084448 gamma Catenin Proteins 0.000 description 2
- 231100000001 growth retardation Toxicity 0.000 description 2
- 201000010536 head and neck cancer Diseases 0.000 description 2
- 208000014829 head and neck neoplasm Diseases 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 102000043380 human ATM Human genes 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 108010008598 insulin-like growth factor binding protein-related protein 1 Proteins 0.000 description 2
- 108010059517 integrin-linked kinase Proteins 0.000 description 2
- 102000007236 involucrin Human genes 0.000 description 2
- 108010033564 involucrin Proteins 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 102100031622 mRNA decay activator protein ZFP36 Human genes 0.000 description 2
- 201000003175 male breast cancer Diseases 0.000 description 2
- 208000010907 male breast carcinoma Diseases 0.000 description 2
- 208000020793 melanoma and neural system tumor syndrome Diseases 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 206010051747 multiple endocrine neoplasia Diseases 0.000 description 2
- 210000000663 muscle cell Anatomy 0.000 description 2
- 201000008026 nephroblastoma Diseases 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 208000002761 neurofibromatosis 2 Diseases 0.000 description 2
- 208000022032 neurofibromatosis type 2 Diseases 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 102000006255 nuclear receptors Human genes 0.000 description 2
- 108020004017 nuclear receptors Proteins 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- ACNHBCIZLNNLRS-UBGQALKQSA-N paxilline Chemical compound N1C2=CC=CC=C2C2=C1[C@]1(C)[C@@]3(C)CC[C@@H]4O[C@H](C(C)(O)C)C(=O)C=C4[C@]3(O)CC[C@H]1C2 ACNHBCIZLNNLRS-UBGQALKQSA-N 0.000 description 2
- 210000002824 peroxisome Anatomy 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 229960000502 poloxamer Drugs 0.000 description 2
- 229920001983 poloxamer Polymers 0.000 description 2
- 108010028075 procathepsin L Proteins 0.000 description 2
- 108090000468 progesterone receptors Proteins 0.000 description 2
- 108010028138 prohibitin Proteins 0.000 description 2
- 102000016670 prohibitin Human genes 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 108010014186 ras Proteins Proteins 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000003196 serial analysis of gene expression Methods 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 210000003932 urinary bladder Anatomy 0.000 description 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 2
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 208000006542 von Hippel-Lindau disease Diseases 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 2
- YMHOBZXQZVXHBM-UHFFFAOYSA-N 2,5-dimethoxy-4-bromophenethylamine Chemical compound COC1=CC(CCN)=C(OC)C=C1Br YMHOBZXQZVXHBM-UHFFFAOYSA-N 0.000 description 1
- AQQSXKSWTNWXKR-UHFFFAOYSA-N 2-(2-phenylphenanthro[9,10-d]imidazol-3-yl)acetic acid Chemical compound C1(=CC=CC=C1)C1=NC2=C(N1CC(=O)O)C1=CC=CC=C1C=1C=CC=CC=12 AQQSXKSWTNWXKR-UHFFFAOYSA-N 0.000 description 1
- 230000035502 ADME Effects 0.000 description 1
- 101150107888 AKT2 gene Proteins 0.000 description 1
- 101150029129 AR gene Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 208000007415 Anhedonia Diseases 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 208000027896 Aortic valve disease Diseases 0.000 description 1
- 208000025490 Apert syndrome Diseases 0.000 description 1
- 108010048907 Arachidonate 15-lipoxygenase Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 102000008056 Aryl Hydrocarbon Receptor Nuclear Translocator Human genes 0.000 description 1
- 108010049386 Aryl Hydrocarbon Receptor Nuclear Translocator Proteins 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 241000282672 Ateles sp. Species 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010003805 Autism Diseases 0.000 description 1
- 208000020706 Autistic disease Diseases 0.000 description 1
- 208000000659 Autoimmune lymphoproliferative syndrome Diseases 0.000 description 1
- 108091005950 Azurite Proteins 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 108700010154 BRCA2 Genes Proteins 0.000 description 1
- 206010062804 Basal cell naevus syndrome Diseases 0.000 description 1
- 101150017888 Bcl2 gene Proteins 0.000 description 1
- 206010004552 Bicuspid aortic valve Diseases 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 101150008921 Brca2 gene Proteins 0.000 description 1
- 102100025399 Breast cancer type 2 susceptibility protein Human genes 0.000 description 1
- 102100036845 C-C motif chemokine 22 Human genes 0.000 description 1
- 108010062802 CD66 antigens Proteins 0.000 description 1
- 108091011896 CSF1 Proteins 0.000 description 1
- 101100492805 Caenorhabditis elegans atm-1 gene Proteins 0.000 description 1
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 201000002927 Cardiofaciocutaneous syndrome Diseases 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- ZEOWTGPWHLSLOG-UHFFFAOYSA-N Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F Chemical compound Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F ZEOWTGPWHLSLOG-UHFFFAOYSA-N 0.000 description 1
- 102000011068 Cdc42 Human genes 0.000 description 1
- 108091007854 Cdh1/Fizzy-related Proteins 0.000 description 1
- 241001515796 Cebinae Species 0.000 description 1
- 102100025051 Cell division control protein 42 homolog Human genes 0.000 description 1
- 101710150820 Cellular tumor antigen p53 Proteins 0.000 description 1
- 108091005944 Cerulean Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241000862448 Chlorocebus Species 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 206010008723 Chondrodystrophy Diseases 0.000 description 1
- 208000037088 Chromosome Breakage Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 208000037463 Chuvash erythrocytosis Diseases 0.000 description 1
- 208000017746 Chuvash polycythemia Diseases 0.000 description 1
- 108091005960 Citrine Proteins 0.000 description 1
- 108090000600 Claudin-1 Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 206010053138 Congenital aplastic anaemia Diseases 0.000 description 1
- 102000002585 Contractile Proteins Human genes 0.000 description 1
- 108010068426 Contractile Proteins Proteins 0.000 description 1
- 206010067380 Costello Syndrome Diseases 0.000 description 1
- 206010066946 Craniofacial dysostosis Diseases 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 201000006526 Crouzon syndrome Diseases 0.000 description 1
- 201000001200 Crouzon syndrome-acanthosis nigricans syndrome Diseases 0.000 description 1
- 108091005943 CyPet Proteins 0.000 description 1
- 108010009356 Cyclin-Dependent Kinase Inhibitor p15 Proteins 0.000 description 1
- 102000009512 Cyclin-Dependent Kinase Inhibitor p15 Human genes 0.000 description 1
- 102000009508 Cyclin-Dependent Kinase Inhibitor p16 Human genes 0.000 description 1
- 108010016788 Cyclin-Dependent Kinase Inhibitor p21 Proteins 0.000 description 1
- 108010016777 Cyclin-Dependent Kinase Inhibitor p27 Proteins 0.000 description 1
- 108010017222 Cyclin-Dependent Kinase Inhibitor p57 Proteins 0.000 description 1
- 102100024462 Cyclin-dependent kinase 4 inhibitor B Human genes 0.000 description 1
- 108010037462 Cyclooxygenase 2 Proteins 0.000 description 1
- 108010072220 Cyclophilin A Proteins 0.000 description 1
- NBSCHQHZLSJFNQ-QTVWNMPRSA-N D-Mannose-6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-QTVWNMPRSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 206010011891 Deafness neurosensory Diseases 0.000 description 1
- 206010070179 Denys-Drash syndrome Diseases 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 208000020401 Depressive disease Diseases 0.000 description 1
- 102100030074 Dickkopf-related protein 1 Human genes 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 108010030483 Dynamin III Proteins 0.000 description 1
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 1
- 102100032257 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 1
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 1
- 101710162545 E3 ubiquitin-protein ligase RNF139 Proteins 0.000 description 1
- 108091005941 EBFP Proteins 0.000 description 1
- 108091005947 EBFP2 Proteins 0.000 description 1
- 108091005942 ECFP Proteins 0.000 description 1
- 101150082819 ERBB3 gene Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010044063 Endocrine-Gland-Derived Vascular Endothelial Growth Factor Proteins 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 206010014967 Ependymoma Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 102000056372 ErbB-3 Receptor Human genes 0.000 description 1
- 241000289659 Erinaceidae Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 101150025764 FGFR3 gene Proteins 0.000 description 1
- 101150082429 FGFR4 gene Proteins 0.000 description 1
- 101150017750 FGFRL1 gene Proteins 0.000 description 1
- 201000004939 Fanconi anemia Diseases 0.000 description 1
- 208000001362 Fetal Growth Retardation Diseases 0.000 description 1
- 102100023600 Fibroblast growth factor receptor 2 Human genes 0.000 description 1
- 101710182389 Fibroblast growth factor receptor 2 Proteins 0.000 description 1
- 102100027842 Fibroblast growth factor receptor 3 Human genes 0.000 description 1
- 101710182396 Fibroblast growth factor receptor 3 Proteins 0.000 description 1
- 102100027844 Fibroblast growth factor receptor 4 Human genes 0.000 description 1
- 102100026149 Fibroblast growth factor receptor-like 1 Human genes 0.000 description 1
- 206010070531 Foetal growth restriction Diseases 0.000 description 1
- 208000007982 Frasier Syndrome Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 230000010337 G2 phase Effects 0.000 description 1
- 102000013446 GTP Phosphohydrolases Human genes 0.000 description 1
- 108091006109 GTPases Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000993347 Gallus gallus Ciliary neurotrophic factor Proteins 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000010956 Glypican Human genes 0.000 description 1
- 108050001154 Glypican Proteins 0.000 description 1
- 102100032530 Glypican-3 Human genes 0.000 description 1
- 108050007237 Glypican-3 Proteins 0.000 description 1
- 208000031995 Gorlin syndrome Diseases 0.000 description 1
- 101710113864 Heat shock protein 90 Proteins 0.000 description 1
- 101710100504 Heat shock protein beta-1 Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 102000006752 Hepatocyte Nuclear Factor 4 Human genes 0.000 description 1
- 208000033640 Hereditary breast cancer Diseases 0.000 description 1
- 206010067943 Hereditary papillary renal carcinoma Diseases 0.000 description 1
- 208000027927 Hereditary papillary renal cell carcinoma Diseases 0.000 description 1
- 108091027305 Heteroduplex Proteins 0.000 description 1
- 101001017818 Homo sapiens ATP-dependent translocase ABCB1 Proteins 0.000 description 1
- 101000775732 Homo sapiens Androgen receptor Proteins 0.000 description 1
- 101000934858 Homo sapiens Breast cancer type 2 susceptibility protein Proteins 0.000 description 1
- 101000823298 Homo sapiens Broad substrate specificity ATP-binding cassette transporter ABCG2 Proteins 0.000 description 1
- 101000713083 Homo sapiens C-C motif chemokine 22 Proteins 0.000 description 1
- 101000984015 Homo sapiens Cadherin-1 Proteins 0.000 description 1
- 101000981093 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 1 Proteins 0.000 description 1
- 101000715398 Homo sapiens Caspase-1 Proteins 0.000 description 1
- 101000867612 Homo sapiens Caspase-2 Proteins 0.000 description 1
- 101000933112 Homo sapiens Caspase-4 Proteins 0.000 description 1
- 101000741087 Homo sapiens Caspase-6 Proteins 0.000 description 1
- 101000741014 Homo sapiens Caspase-7 Proteins 0.000 description 1
- 101000983523 Homo sapiens Caspase-9 Proteins 0.000 description 1
- 101000934426 Homo sapiens Cell division control protein 42 homolog Proteins 0.000 description 1
- 101000749331 Homo sapiens Claudin-1 Proteins 0.000 description 1
- 101000980919 Homo sapiens Cyclin-dependent kinase 4 inhibitor B Proteins 0.000 description 1
- 101000944380 Homo sapiens Cyclin-dependent kinase inhibitor 1 Proteins 0.000 description 1
- 101000944361 Homo sapiens Cyclin-dependent kinase inhibitor 1B Proteins 0.000 description 1
- 101000944365 Homo sapiens Cyclin-dependent kinase inhibitor 1C Proteins 0.000 description 1
- 101000980932 Homo sapiens Cyclin-dependent kinase inhibitor 2A Proteins 0.000 description 1
- 101000864646 Homo sapiens Dickkopf-related protein 1 Proteins 0.000 description 1
- 101000817599 Homo sapiens Dynamin-3 Proteins 0.000 description 1
- 101001015963 Homo sapiens E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 1
- 101000917134 Homo sapiens Fibroblast growth factor receptor 4 Proteins 0.000 description 1
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 description 1
- 101001036709 Homo sapiens Heat shock protein beta-1 Proteins 0.000 description 1
- 101000962530 Homo sapiens Hyaluronidase-1 Proteins 0.000 description 1
- 101000962526 Homo sapiens Hyaluronidase-2 Proteins 0.000 description 1
- 101001041128 Homo sapiens Hyaluronidase-3 Proteins 0.000 description 1
- 101000852815 Homo sapiens Insulin receptor Proteins 0.000 description 1
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 1
- 101000853009 Homo sapiens Interleukin-24 Proteins 0.000 description 1
- 101000998139 Homo sapiens Interleukin-32 Proteins 0.000 description 1
- 101000614436 Homo sapiens Keratin, type I cytoskeletal 14 Proteins 0.000 description 1
- 101001046960 Homo sapiens Keratin, type II cytoskeletal 1 Proteins 0.000 description 1
- 101000716729 Homo sapiens Kit ligand Proteins 0.000 description 1
- 101000916628 Homo sapiens Macrophage colony-stimulating factor 1 Proteins 0.000 description 1
- 101000582631 Homo sapiens Menin Proteins 0.000 description 1
- 101000981728 Homo sapiens Myeloid differentiation primary response protein MyD88 Proteins 0.000 description 1
- 101001000104 Homo sapiens Myosin-11 Proteins 0.000 description 1
- 101000979629 Homo sapiens Nucleoside diphosphate kinase A Proteins 0.000 description 1
- 101000601724 Homo sapiens Paired box protein Pax-5 Proteins 0.000 description 1
- 101000735484 Homo sapiens Paired box protein Pax-9 Proteins 0.000 description 1
- 101001067833 Homo sapiens Peptidyl-prolyl cis-trans isomerase A Proteins 0.000 description 1
- 101000579484 Homo sapiens Period circadian protein homolog 1 Proteins 0.000 description 1
- 101000741790 Homo sapiens Peroxisome proliferator-activated receptor gamma Proteins 0.000 description 1
- 101000578474 Homo sapiens Polyunsaturated fatty acid lipoxygenase ALOX15B Proteins 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- 101000610537 Homo sapiens Prokineticin-1 Proteins 0.000 description 1
- 101000605127 Homo sapiens Prostaglandin G/H synthase 2 Proteins 0.000 description 1
- 101000585703 Homo sapiens Protein L-Myc Proteins 0.000 description 1
- 101000685726 Homo sapiens Protein S100-A2 Proteins 0.000 description 1
- 101001000998 Homo sapiens Protein phosphatase 1 regulatory subunit 12C Proteins 0.000 description 1
- 101000920560 Homo sapiens Putative endoplasmin-like protein Proteins 0.000 description 1
- 101000708222 Homo sapiens Ras and Rab interactor 2 Proteins 0.000 description 1
- 101000712958 Homo sapiens Ras association domain-containing protein 1 Proteins 0.000 description 1
- 101000712972 Homo sapiens Ras association domain-containing protein 4 Proteins 0.000 description 1
- 101000727472 Homo sapiens Reticulon-4 Proteins 0.000 description 1
- 101000709370 Homo sapiens S-phase kinase-associated protein 2 Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 101000623857 Homo sapiens Serine/threonine-protein kinase mTOR Proteins 0.000 description 1
- 101000701928 Homo sapiens Serpin B5 Proteins 0.000 description 1
- 101000826373 Homo sapiens Signal transducer and activator of transcription 3 Proteins 0.000 description 1
- 101000651893 Homo sapiens Slit homolog 3 protein Proteins 0.000 description 1
- 101000708614 Homo sapiens Smoothened homolog Proteins 0.000 description 1
- 101000693900 Homo sapiens Sodium-coupled monocarboxylate transporter 1 Proteins 0.000 description 1
- 101000685678 Homo sapiens Solute carrier family 22 member 18 Proteins 0.000 description 1
- 101000706156 Homo sapiens Syntaxin-11 Proteins 0.000 description 1
- 101000653663 Homo sapiens T-complex protein 1 subunit epsilon Proteins 0.000 description 1
- 101000712669 Homo sapiens TGF-beta receptor type-2 Proteins 0.000 description 1
- 101000626142 Homo sapiens Tensin-1 Proteins 0.000 description 1
- 101000626155 Homo sapiens Tensin-4 Proteins 0.000 description 1
- 101000658608 Homo sapiens Tetraspanin-32 Proteins 0.000 description 1
- 101000659879 Homo sapiens Thrombospondin-1 Proteins 0.000 description 1
- 101000733249 Homo sapiens Tumor suppressor ARF Proteins 0.000 description 1
- 101000606090 Homo sapiens Tyrosinase Proteins 0.000 description 1
- 101000643908 Homo sapiens Ubiquitin carboxyl-terminal hydrolase 7 Proteins 0.000 description 1
- 101000777301 Homo sapiens Uteroglobin Proteins 0.000 description 1
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 description 1
- 101001074035 Homo sapiens Zinc finger protein GLI2 Proteins 0.000 description 1
- 101150065069 Hsp90b1 gene Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 241000701806 Human papillomavirus Species 0.000 description 1
- 101710199679 Hyaluronidase-1 Proteins 0.000 description 1
- 101710199674 Hyaluronidase-2 Proteins 0.000 description 1
- 108050004076 Hyaluronidase-3 Proteins 0.000 description 1
- 206010020608 Hypercoagulation Diseases 0.000 description 1
- 201000005503 Hypoplastic left heart syndrome Diseases 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 1
- 102000038455 IGF Type 1 Receptor Human genes 0.000 description 1
- 108010031794 IGF Type 1 Receptor Proteins 0.000 description 1
- 102000038460 IGF Type 2 Receptor Human genes 0.000 description 1
- 108010031792 IGF Type 2 Receptor Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108010001127 Insulin Receptor Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 101710181615 Interleukin-32 Proteins 0.000 description 1
- 102100026019 Interleukin-6 Human genes 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 208000009289 Jackson-Weiss syndrome Diseases 0.000 description 1
- 108700003486 Jagged-1 Proteins 0.000 description 1
- 206010023201 Joint contracture Diseases 0.000 description 1
- 201000007493 Kallmann syndrome Diseases 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 108010070514 Keratin-1 Proteins 0.000 description 1
- 108010066321 Keratin-14 Proteins 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000282838 Lama Species 0.000 description 1
- 241000288903 Lemuridae Species 0.000 description 1
- 208000035036 Lethal congenital contracture syndrome type 2 Diseases 0.000 description 1
- 208000035752 Live birth Diseases 0.000 description 1
- 201000005027 Lynch syndrome Diseases 0.000 description 1
- 201000001597 Lynch syndrome 1 Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108091008065 MIR21 Proteins 0.000 description 1
- 101150053046 MYD88 gene Proteins 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 108010047230 Member 1 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 description 1
- 108010090306 Member 2 Subfamily G ATP Binding Cassette Transporter Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 1
- 206010068320 Microencephaly Diseases 0.000 description 1
- 108091062154 Mir-205 Proteins 0.000 description 1
- 238000012347 Morris Water Maze Methods 0.000 description 1
- 101150097381 Mtor gene Proteins 0.000 description 1
- 208000007326 Muenke Syndrome Diseases 0.000 description 1
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- 102100036639 Myosin-11 Human genes 0.000 description 1
- 108010081372 NM23 Nucleoside Diphosphate Kinases Proteins 0.000 description 1
- 102000005238 NM23 Nucleoside Diphosphate Kinases Human genes 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 208000009869 Neu-Laxova syndrome Diseases 0.000 description 1
- 102000014413 Neuregulin Human genes 0.000 description 1
- 108050003475 Neuregulin Proteins 0.000 description 1
- 101800000675 Neuregulin-2 Proteins 0.000 description 1
- 208000003019 Neurofibromatosis 1 Diseases 0.000 description 1
- 208000024834 Neurofibromatosis type 1 Diseases 0.000 description 1
- 108010085839 Neurofibromin 2 Proteins 0.000 description 1
- 102000007517 Neurofibromin 2 Human genes 0.000 description 1
- 108010077641 Nogo Proteins Proteins 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 206010029748 Noonan syndrome Diseases 0.000 description 1
- 102000001759 Notch1 Receptor Human genes 0.000 description 1
- 102100023252 Nucleoside diphosphate kinase A Human genes 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 206010053142 Olfacto genital dysplasia Diseases 0.000 description 1
- 201000010133 Oligodendroglioma Diseases 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108010049358 Oncogene Protein p65(gag-jun) Proteins 0.000 description 1
- 108010058765 Oncogene Protein pp60(v-src) Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010077077 Osteonectin Proteins 0.000 description 1
- 102000009890 Osteonectin Human genes 0.000 description 1
- 108010045055 PAX5 Transcription Factor Proteins 0.000 description 1
- 108010067946 PAX9 Transcription Factor Proteins 0.000 description 1
- 102000016828 PAX9 Transcription Factor Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000023984 PPAR alpha Human genes 0.000 description 1
- 108010028924 PPAR alpha Proteins 0.000 description 1
- 102000000536 PPAR gamma Human genes 0.000 description 1
- 208000013609 PPARG-related familial partial lipodystrophy Diseases 0.000 description 1
- 101150073900 PTEN gene Proteins 0.000 description 1
- 102100034901 Paired box protein Pax-9 Human genes 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 101710111198 Peptidyl-prolyl cis-trans isomerase A Proteins 0.000 description 1
- 102100028293 Period circadian protein homolog 1 Human genes 0.000 description 1
- 206010034764 Peutz-Jeghers syndrome Diseases 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 101710132081 Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN Proteins 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 108010064218 Poly (ADP-Ribose) Polymerase-1 Proteins 0.000 description 1
- 102100023712 Poly [ADP-ribose] polymerase 1 Human genes 0.000 description 1
- 102000012338 Poly(ADP-ribose) Polymerases Human genes 0.000 description 1
- 108010061844 Poly(ADP-ribose) Polymerases Proteins 0.000 description 1
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 1
- 208000008601 Polycythemia Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102100031950 Polyunsaturated fatty acid lipoxygenase ALOX15 Human genes 0.000 description 1
- 102100027921 Polyunsaturated fatty acid lipoxygenase ALOX15B Human genes 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 102100022668 Pro-neuregulin-2, membrane-bound isoform Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108091008611 Protein Kinase B Proteins 0.000 description 1
- 102100030128 Protein L-Myc Human genes 0.000 description 1
- 101710156962 Protein S100-A2 Proteins 0.000 description 1
- 102100032702 Protein jagged-1 Human genes 0.000 description 1
- 108700037966 Protein jagged-1 Proteins 0.000 description 1
- 102100032733 Protein jagged-2 Human genes 0.000 description 1
- 101710170213 Protein jagged-2 Proteins 0.000 description 1
- 102100035620 Protein phosphatase 1 regulatory subunit 12C Human genes 0.000 description 1
- 208000014841 Proteus-like syndrome Diseases 0.000 description 1
- 102000013535 Proto-Oncogene Proteins c-bcl-2 Human genes 0.000 description 1
- 108010090931 Proto-Oncogene Proteins c-bcl-2 Proteins 0.000 description 1
- 102100031916 Putative endoplasmin-like protein Human genes 0.000 description 1
- 108010010469 Qa-SNARE Proteins Proteins 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 108091007364 RNF139 Proteins 0.000 description 1
- 102100031490 Ras and Rab interactor 2 Human genes 0.000 description 1
- 102100033243 Ras association domain-containing protein 1 Human genes 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108010055623 S-Phase Kinase-Associated Proteins Proteins 0.000 description 1
- 201000001079 SADDAN Diseases 0.000 description 1
- 108010005173 SERPIN-B5 Proteins 0.000 description 1
- 108091006750 SLC22A18 Proteins 0.000 description 1
- 108091006274 SLC5A8 Proteins 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 101001025539 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Homothallic switching endonuclease Proteins 0.000 description 1
- 241000288961 Saguinus imperator Species 0.000 description 1
- 241000282695 Saimiri Species 0.000 description 1
- 241000235346 Schizosaccharomyces Species 0.000 description 1
- 208000009966 Sensorineural Hearing Loss Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 208000017601 Severe achondroplasia-developmental delay-acanthosis nigricans syndrome Diseases 0.000 description 1
- 206010062282 Silver-Russell syndrome Diseases 0.000 description 1
- 206010072610 Skeletal dysplasia Diseases 0.000 description 1
- 101710090597 Smoothened homolog Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 108010039445 Stem Cell Factor Proteins 0.000 description 1
- 108010087999 Steryl-Sulfatase Proteins 0.000 description 1
- 102100038021 Steryl-sulfatase Human genes 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 101710100613 Tensin-1 Proteins 0.000 description 1
- 101710100614 Tensin-4 Proteins 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 101710132098 Tetraspanin-32 Proteins 0.000 description 1
- 108010046722 Thrombospondin 1 Proteins 0.000 description 1
- 102000005747 Transcription Factor RelA Human genes 0.000 description 1
- 108010031154 Transcription Factor RelA Proteins 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 241001325280 Tricardia watsonii Species 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 101150020913 USP7 gene Proteins 0.000 description 1
- 102000003431 Ubiquitin-Conjugating Enzyme Human genes 0.000 description 1
- 108060008747 Ubiquitin-Conjugating Enzyme Proteins 0.000 description 1
- 108700011958 Ubiquitin-Specific Peptidase 7 Proteins 0.000 description 1
- 229940126752 Ubiquitin-specific protease 7 inhibitor Drugs 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 102100031083 Uteroglobin Human genes 0.000 description 1
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 1
- 241000545067 Venus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 101150046474 Vhl gene Proteins 0.000 description 1
- 241001416177 Vicugna pacos Species 0.000 description 1
- 208000006269 X-Linked Bulbo-Spinal Atrophy Diseases 0.000 description 1
- 241000269368 Xenopus laevis Species 0.000 description 1
- 108010088665 Zinc Finger Protein Gli2 Proteins 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 102000009120 alpha Subunit Hypoxia-Inducible Factor 1 Human genes 0.000 description 1
- 108010048418 alpha Subunit Hypoxia-Inducible Factor 1 Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 102000001307 androgen receptors Human genes 0.000 description 1
- 206010068168 androgenetic alopecia Diseases 0.000 description 1
- 201000002996 androgenic alopecia Diseases 0.000 description 1
- 229940030486 androgens Drugs 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 239000003524 antilipemic agent Substances 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 210000001765 aortic valve Anatomy 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 101150051494 atr gene Proteins 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 208000021654 bicuspid aortic valve disease Diseases 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 108091005948 blue fluorescent proteins Proteins 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 108010051348 cdc42 GTP-Binding Protein Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 208000025997 central nervous system neoplasm Diseases 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 201000002687 childhood acute myeloid leukemia Diseases 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000011035 citrine Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000030944 contact inhibition Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 108010082025 cyan fluorescent protein Proteins 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000021953 cytokinesis Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000447 dimerizing effect Effects 0.000 description 1
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 208000014616 embryonal neoplasm Diseases 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 208000024519 eye neoplasm Diseases 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 201000004292 familial erythrocytosis 2 Diseases 0.000 description 1
- 201000000497 familial melanoma Diseases 0.000 description 1
- 201000002086 familial partial lipodystrophy type 3 Diseases 0.000 description 1
- 201000005306 familial renal papillary carcinoma Diseases 0.000 description 1
- 208000030941 fetal growth restriction Diseases 0.000 description 1
- 210000000604 fetal stem cell Anatomy 0.000 description 1
- 101150016624 fgfr1 gene Proteins 0.000 description 1
- 101150088071 fgfr2 gene Proteins 0.000 description 1
- 238000012048 forced swim test Methods 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 208000012446 glioma susceptibility Diseases 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- 230000010005 growth-factor like effect Effects 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 201000002222 hemangioblastoma Diseases 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 108091008634 hepatocyte nuclear factors 4 Proteins 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 208000025581 hereditary breast carcinoma Diseases 0.000 description 1
- 208000036427 hereditary nonpolyposis type 2 colorectal cancer Diseases 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 102000045609 human NOTCH1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 201000010072 hypochondroplasia Diseases 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000000530 impalefection Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 108090000237 interleukin-24 Proteins 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000006662 intracellular pathway Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 201000008632 juvenile polyposis syndrome Diseases 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 201000004803 lethal congenital contracture syndrome 2 Diseases 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000003199 leukotriene receptor blocking agent Substances 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007787 long-term memory Effects 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 108091005949 mKalama1 Proteins 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 201000010893 malignant breast melanoma Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 108091029119 miR-34a stem-loop Proteins 0.000 description 1
- 108091079013 miR-34b Proteins 0.000 description 1
- 108091084018 miR-34b stem-loop Proteins 0.000 description 1
- 108091063470 miR-34b-1 stem-loop Proteins 0.000 description 1
- 108091049916 miR-34b-2 stem-loop Proteins 0.000 description 1
- 108091057222 miR-34b-3 stem-loop Proteins 0.000 description 1
- 108091092639 miR-34b-4 stem-loop Proteins 0.000 description 1
- 208000004141 microcephaly Diseases 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 208000022499 mismatch repair cancer syndrome Diseases 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 230000007659 motor function Effects 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000002464 muscle smooth vascular Anatomy 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 230000018341 negative regulation of fibrinolysis Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000006576 neuronal survival Effects 0.000 description 1
- 201000005734 nevoid basal cell carcinoma syndrome Diseases 0.000 description 1
- 230000007959 normoxia Effects 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 201000008106 ocular cancer Diseases 0.000 description 1
- 102000027450 oncoproteins Human genes 0.000 description 1
- 108091008819 oncoproteins Proteins 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 208000020037 osteoglophonic dysplasia Diseases 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 210000002990 parathyroid gland Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000011340 peptidyl-tyrosine autophosphorylation Effects 0.000 description 1
- 239000003614 peroxisome proliferator Substances 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 230000001402 polyadenylating effect Effects 0.000 description 1
- 201000008519 polycystic kidney disease 1 Diseases 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 230000009596 postnatal growth Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000006977 prepulse inhibition Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000861 pro-apoptotic effect Effects 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 208000023958 prostate neoplasm Diseases 0.000 description 1
- 230000009993 protective function Effects 0.000 description 1
- 230000003331 prothrombotic effect Effects 0.000 description 1
- 238000013114 radial arm maze test Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000021014 regulation of cell growth Effects 0.000 description 1
- 230000001718 repressive effect Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 238000010825 rotarod performance test Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 230000036362 sensorimotor function Effects 0.000 description 1
- 208000023573 sensorineural hearing loss disease Diseases 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000037436 splice-site mutation Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000032270 susceptibility to 1 ovarian cancer Diseases 0.000 description 1
- 208000020792 susceptibility to uveal melanoma Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 208000011317 telomere syndrome Diseases 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 201000003896 thanatophoric dysplasia Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical group [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 201000005665 thrombophilia Diseases 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 210000000515 tooth Anatomy 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 101150023860 tpr gene Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- GWBUNZLLLLDXMD-UHFFFAOYSA-H tricopper;dicarbonate;dihydroxide Chemical compound [OH-].[OH-].[Cu+2].[Cu+2].[Cu+2].[O-]C([O-])=O.[O-]C([O-])=O GWBUNZLLLLDXMD-UHFFFAOYSA-H 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000010396 two-hybrid screening Methods 0.000 description 1
- 210000002444 unipotent stem cell Anatomy 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 239000000277 virosome Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
Definitions
- the invention generally relates to genetically modified animals or cells comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression.
- the invention relates to the use of a zinc finger nuclease-mediated process to edit chromosomal sequences encoding proteins involved in tumor suppression.
- Tumor suppressors help regulate cell division and cell death. Mutations in tumor suppressor genes can lead to abnormal cell division and uncontrolled cell division, which is responsible for many types of cancer. This influx of cells in the body can grow into tissues, leading to specific cancers associated with various organs. Additionally, uncontrolled cell growth is responsible for blood cancer, such as leukemia. Half of all men and one-third of all women in the United States will develop cancer during their lifetimes. While research is being done to develop with new treatments for cancer, new treatment strategies and therapies are needed.
- mice that are mutated for the genes involved in tumor suppression processes, including knockouts, multiple mutant lines (double knockouts, triple knockouts, etc.) and/or over-expression of alleles that either cause disease or are associated with disease in humans, as well as “humanized” animals that express or over-express human homologues of relevant genes in animals. Such animals could serve as research tools to develop and/or test new treatments for cancer.
- One aspect of the present disclosure encompasses a genetically modified animal comprising at least one edited chromosomal sequence encoding a sequence involved in tumor suppression.
- a further aspect provides a non-human embryo comprising at least one RNA molecule encoding a zinc finger nuclease that recognizes a chromosomal sequence encoding a protein involved in tumor suppression, and, optionally, at least one donor polynucleotide comprising a sequence encoding a protein involved in tumor suppression.
- Another aspect provides a genetically modified cell comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression.
- An alternate aspect provides a zinc finger nuclease comprising (a) a zinc finger DNA binding domain that binds a sequence having at least about 80% sequence identity to a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8; and (b) a cleavage domain.
- a further aspect provides a nucleic acid sequence recognized by a zinc finger nuclease.
- the nucleic acid sequence has at least about 80% sequence identity to a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8.
- Yet another aspect encompasses a method for assessing the therapeutic effect and/or toxicity of an agent.
- the method comprises (a) contacting a first genetically modified animal comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppressor with the agent; (b) measuring a therapeutic and/or toxicity response in the first animal; and (c) comparing the response in (b) to results obtained from a second genetically modified animal comprising the same edited chromosomal sequence encoding a protein involved in tumor suppression, wherein the second animal is not contacted with the agent.
- FIG. 1 illustrates editing of the p53 locus in rats.
- a Cel-1 assay in which the presence of cleavage products indicated editing of the p53 gene.
- FIG. 2 illustrates knockout of the p53 gene in rats.
- FIG. 3 presents the DNA sequences of edited BCRP loci in two animals.
- A Shows a region of the rat BCRP locus (SEQ ID NO:1) comprising a 588 bp deletion in exon 7.
- B Presents a region of the rat BCRP locus (SEQ ID NO:2) comprising a 696 bp deletion in exon 7. The exon sequence is shown in green; the target site is presented in yellow, and the deletions are shown in dark blue.
- the present disclosure provides a genetically modified animal or animal cell comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression.
- the edited chromosomal sequence may be (1) inactivated, (2) modified, or (3) comprise an integrated sequence.
- An inactivated chromosomal sequence is altered such that a functional protein is not made.
- a genetically modified animal comprising an inactivated chromosomal sequence may be termed a “knock-out” or a “conditional knock-out.”
- a genetically modified animal comprising an integrated sequence may be termed a “knock-in” or a “conditional knock-in.”
- a knock-in animal may be a humanized animal.
- a genetically modified animal comprising a modified chromosomal sequence may comprise a targeted point mutation(s) or other modification such that an altered protein product is produced.
- the chromosomal sequence encoding a protein involved in tumor suppression generally is edited using a zinc finger nuclease-mediated process. Briefly, the process comprises introducing into an embryo or cell at least one RNA molecule encoding a targeted zinc finger nuclease and, optionally, at least one accessory polynucleotide.
- the method further comprises incubating the embryo or cell to allow expression of the zinc finger nuclease, wherein a double-stranded break introduced into the targeted chromosomal sequence by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process or a homology-directed DNA repair process.
- the method of editing chromosomal sequences involved in tumor suppression using targeted zinc finger nuclease technology is rapid, precise, and highly efficient.
- One aspect of the present disclosure provides a genetically modified animal in which at least one chromosomal sequence associated with tumor suppression has been edited.
- the edited chromosomal sequence may be inactivated such that the sequence is not transcribed and/or a functional protein is not produced.
- the chromosomal sequence may be edited such that the regulation of expression of the protein is altered.
- the chromosomal sequence may be modified such that the protein associated with tumor suppression is over-produced.
- the edited chromosomal sequence may also be modified such that it codes for an altered tumor suppressor protein.
- the chromosomal sequence may be modified such that at least one nucleotide is changed and the expressed protein comprises at least one changed amino acid residue (i.e., comprises a missense mutation).
- the edited chromosomal sequence may comprise a chromosomally integrated sequence encoding a protein associated with tumor suppression.
- the chromosomally integrated sequence may encode an endogenous protein associated with tumor suppression normally found in the animal, or the integrated sequence may encode an exogenous orthologous protein associated with tumor suppression, or combinations of both.
- the genetically modified animal disclosed herein may be heterozygous for the edited chromosomal sequence.
- the genetically modified animal may be homozygous for the edited chromosomal sequence.
- the genetically modified animal may comprise at least one inactivated chromosomal sequence encoding a protein associated with tumor suppression.
- the inactivated chromosomal sequence may include a deletion mutation (i.e., deletion of one or more nucleotides), an insertion mutation (i.e., insertion of one or more nucleotides), or a point mutation (i.e., substitution of a single nucleotide for another nucleotide).
- the deletion, insertion, or point mutation may lead to frame shift and/or splice site mutations such that at least one premature stop codon is introduced. As a consequence of the mutation, the targeted chromosomal sequence is inactivated and a functional protein is not produced.
- the inactivated chromosomal sequence comprises no exogenously introduced sequence. Such an animal may be termed a “knock-out.” Also included herein are genetically modified animals in which two, three, or more chromosomal sequences encoding proteins associated with tumor suppression are inactivated.
- the edited chromosomal sequence may be modified such that it codes for an altered protein associated with tumor suppression.
- the chromosomal sequence may be modified such that at least one nucleotide is changed and the expressed protein comprises at least one changed amino acid residue (i.e., a missense mutation).
- the chromosomal sequence may be modified to comprise more than one missense mutation such that more than one amino acid is changed.
- the chromosomal sequence may be modified to have a three nucleotide deletion or insertion such that the expressed protein comprises a single amino acid deletion or insertion, provided such a protein is functional.
- the modified protein associated with tumor suppression may have altered substrate specificity, altered enzyme activity, altered kinetic rates, and so forth.
- the modified protein comprises at least one modification such that the altered version of the protein provides tumor suppression activity.
- the modified protein comprises at least one modification such that the altered version of the protein attenuates tumor suppression.
- the genetically modified animal may comprise at least one chromosomally integrated sequence encoding a protein associated with tumor suppression. Integration of the sequence encoding a protein associated with tumor suppression may be random or it may be targeted.
- an exogenous sequence encoding an orthologous or an endogenous protein associated with tumor suppression may be integrated into a chromosomal sequence encoding a protein associated with tumor suppression (or another protein) such that the endogenous chromosomal sequence is inactivated, but wherein the exogenous sequence encoding the orthologous or endogenous protein may be expressed.
- the sequence encoding the orthologous or endogenous protein may be operably linked to an endogenous promoter control sequence.
- the promoter control sequence may be native to the exogenous sequence or the exogenous sequence may be operably linked to a heterologous promoter control sequence.
- the exogenous sequence encoding the orthologous or endogenous protein may be such that the protein associated with tumor suppression is over-produced, or the tissue-specific or temporal expression of the protein is altered, or a combination thereof.
- an exogenous sequence encoding an orthologous or endogenous protein may be integrated into a chromosomal sequence without affecting expression of an endogenous chromosomal sequence.
- an exogenous sequence encoding a protein associated with tumor suppression may be integrated into a “safe harbor” locus, such as the Rosa26 locus, HPRT locus, or AAVS1 locus, wherein the exogenous sequence encoding the orthologous or endogenous protein may be expressed or over-expressed.
- a “safe harbor” locus such as the Rosa26 locus, HPRT locus, or AAVS1 locus
- An animal comprising a chromosomally integrated sequence encoding a protein associated with tumor suppression may be called a “knock-in,” and it should be understood that in such an iteration of the animal, no selectable marker is present.
- the sequence encoding a protein associated with tumor suppression also may be modified to include a tag or reporter.
- Suitable reporters include selectable markers such as cloramphenicol acetyltransferase (CAT) and neomycin phosphotransferase (neo), and fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein, or any genetically engineered variant thereof that improves the reporter performance.
- selectable markers such as cloramphenicol acetyltransferase (CAT) and neomycin phosphotransferase (neo)
- fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein, or any genetically engineered variant thereof that improves the reporter performance.
- Non-limiting examples of known such FP variants include EGFP, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet).
- the chromosomally integrated sequence encoding a protein associated with tumor suppression may encode the wild-type form of the protein.
- the chromosomally integrated sequence encoding a protein associated with tumor suppression may comprise at least one modification such that an altered version of the protein is produced.
- the chromosomally integrated sequence encoding a protein associated with tumor suppression comprises at least one modification such that the altered version of the protein enhances tumor suppression.
- the chromosomally integrated sequence encoding a protein associated with tumor suppression comprises at least one modification such that the altered version of the protein attenuates tumor suppression.
- the genetically modified animal may be a “humanized” animal comprising at least one chromosomally integrated sequence encoding a functional tumor suppressor protein.
- the functional human protein may have no corresponding ortholog in the genetically modified animal.
- the wild-type animal from which the genetically modified animal is derived may comprise an ortholog corresponding to the human tumor suppressor protein.
- the orthologous sequence in the “humanized” animal is inactivated such that no functional protein is made and the “humanized” animal comprises at least one chromosomally integrated sequence encoding the human tumor suppressor protein.
- a humanized animal may comprise an inactivated p53 sequence and a chromosomally integrated sequence encoding human p53 protein.
- “humanized” animals may be generated by crossing a knock-out animal with a knock-in animal comprising the chromosomally integrated sequence.
- the genetically modified animal may comprise at least one edited chromosomal sequence encoding a protein associated with tumor suppression such that the expression pattern of the protein associated with tumor suppression is altered.
- regulatory regions controlling the expression of the protein such as a promoter or transcription binding site, may be altered such that the protein associated with tumor suppression is over-produced, or the tissue-specific or temporal expression of the protein is altered, or a combination thereof.
- the expression pattern of the protein associated with tumor suppression may be altered using a conditional knockout system.
- a non-limiting example of a conditional knockout system includes a Cre-lox recombination system.
- a Cre-lox recombination system comprises a Cre recombinase enzyme, a site-specific DNA recombinase that can catalyze the recombination of a nucleic acid sequence between specific sites (lox sites) in a nucleic acid molecule.
- Methods of using this system to produce temporal and tissue specific expression are known in the art.
- a genetically modified animal is generated with lox sites flanking a chromosomal sequence, such as a chromosomal sequence encoding a protein associated with tumor suppression.
- the genetically modified animal comprising the lox-flanked chromosomal sequence encoding a protein associated with tumor suppression may then be crossed with another genetically modified animal expressing Cre recombinase.
- Progeny animals comprising the lox-flanked chromosomal sequence and the Cre recombinase are then produced, and the lox-flanked chromosomal sequence encoding a protein associated with tumor suppression is recombined, leading to deletion or inversion of the chromosomal sequence encoding a protein associated with tumor suppression.
- Expression of Cre recombinase may be temporally and conditionally regulated to effect temporally and conditionally regulated recombination of the chromosomal sequence encoding a protein associated with tumor suppression.
- Tumor suppression genes are genes whose protein products protect a cell from one step on the path to cancer.
- a mutation in a tumor suppressor gene may cause a loss or reduction in the protective function of its protein product, thereby increasing the probability that a tumor will form, leading to cancer, usually in combination with other genetic changes.
- the proteins encoded by tumor suppressor genes have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes both.
- Tumor suppressor proteins are involved in the repression of genes essential for the continuing cell cycle; coupling the cell cycle to DNA damage so that the cell cycle can continue; initiating apoptosis in the cell if the damage cannot be repaired; and cell adhesion to prevent tumors from dispersing, blocking a loss of contact inhibition, and inhibiting metastasis.
- Mutations in tumor suppressor genes can lead to various types of cancer, including but not limited to Retinoblastoma, Human Papilloma Virus, Wilms Tumor, Neurofibromatosis Type 1, Neurofibromatosis Type 2, familial adenomatous polyposis, Colon Cancer, Von Hippel-Lindau syndrome, Li-Fraumeni Syndrome, Familial Juvenile Polyposis syndrome, Familial Breast Cancer, Cowden Syndrome, Koz-Jeghers Syndrome, Hereditary Nonpolyposis Colon Cancer Type 1, Hereditary Nonpolyposis Colon Cancer Type 2, Familial diffuse-type Gastic Cancer, Familial Melanoma, Gorlin Syndrome, Multiple Endocrine Neoplasia Type 1, and other tumor-related diseases.
- the present disclosure comprises editing of any chromosomal sequences that encodes a protein associated with tumor suppression.
- the proteins associated with tumor suppression are typically selected based on an experimental association of the protein of interest with a cancer. For example, the production rate or circulating concentration of a protein associated with tumor suppression may be elevated or depressed in a population having cancer relative to a population not having cancer. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry.
- proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry.
- the proteins associated with tumor suppression may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).
- genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).
- proteins involved in tumor suppression and their encoding chromosomal sequences may comprise, but is not limited to, TNF (tumor necrosis factor (TNF superfamily, member 2)), TP53 (tumor protein p53), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)), FN1 (fibronectin 1), TSC1 (tuberous sclerosis 1), PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), PTEN (phosphatase and tensin homolog), PCNA (proliferating cell nuclear antigen), COL18A1 (collagen, type XVIII, alpha 1), TSSC4 (tumor suppressing subtransferable candidate 4), JUN (jun oncogene), MAPK8 (mitogen-
- BTRC beta-transducin repeat containing
- NKX3-1 NK3 homeobox 1
- GPC3 glypican 3
- CREB3 cAMP responsive element binding protein 3
- PLCB3 phospholipase C, beta 3 (phosphatidylinositol-specific)
- DMPK distrophia myotonica-protein kinase
- BLNK B-cell linker
- PPIA peptidylprolyl isomerase A (cyclophilin A)
- DAB2 disabled homolog 2, mitogen-responsive phosphoprotein ( Drosophila )
- KLF4 Kruppel-like factor 4 (gut)
- RUNX3 runt-related transcription factor 3
- FLG filaggrin
- IVL involucrin
- COTS chaperonin containing TCP1, subunit 5 (epsilon)
- LRPAP1 low density lipoprotein receptor-related protein associated protein 1
- IGF2R IGF2
- tumor suppression proteins include ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor 1a), HIF3a (hypoxia), HIF3a (
- ATM or ataxia telangiectasia mutated, provides instructions for making a protein, primarily located in the nucleus, which helps control the rate at which cells grow and divide. Additionally, ATM plays an important role in the normal development and activity of several body systems, including the nervous system and the immune system. ATM protein assists cells in recognizing damaged or broken DNA strands and coordinates DNA repair by activating enzymes that repair breaks in the DNA. The stability of the cell's genetic information is maintained by the efficient repair of damaged DNA. The ATM protein is of great interest in cancer research because of its central role in cell division and DNA repair.
- Ataxia-telangiectasia is caused by one of several hundred mutations in the ATM gene. Those afflicted with this disorder have mutations in both copies of the ATM gene in a cell.
- Breast cancer has also been found to be associated with the ATM gene. Patients with at least one family member with ataxia-telangiectasia are thought to have an increased risk of developing breast cancer.
- the mutation in the ATM gene prevents many of the body's cells from correctly repairing damaged DNA. People who have only one copy of the ATM gene in each cell, due to a deletion, are also at an increased risk of developing breast cancer.
- ATR or ataxia telangiectasia and Rad3 related, is a protein kinase. Mutations of the ATR gene are associated with Sekel syndrome, a rare autosomal recessive disorder characterized by growth retardation, microencephaly with mental retardation, and a characteristic “bird headed” facial appearance. ATR is thought to be involved in DNA replication and DNA repair.
- EGFR or epidermal growth factor receptor is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family.
- EGFR is a cell surface protein that binds to epidermal growth factor. When EGFR binds to a ligand, receptor dimerization and tyrosine autophosphorylation is induced, leading to cell proliferation. Mutations in EGFR are associated with lung cancer.
- ERBB2 or v-erb-b2 erythroblastic leukemia viral oncogene homolog 2
- Her-2/neu ERBB2 growth factor receptor
- ERBB2 growth factor receptor is located on the cell surface, where it associates with similar receptors to form a complex. Growth factors bind to these similar receptors and trigger the receptor complex to relay signals inside the cell activating certain genes that promote cell growth. It is thought that ERBB2 plays a role in cell adhesion, cell specialization, and cell movement.
- Breast cancer is associated with the ERBB2 gene and amplification of the ERBB2 gene is found in about 25% of breast cancers.
- the mutations in the ERBB2 gene are somatic mutations, thus, they are not inherited.
- One mechanism of somatic mutations is where DNA replicates in preparation for cell division resulting in multiple copies of the gene on a chromosome. Tumors can form as a result of multiple gene copies.
- Amplification of ERBB2 has also been linked to other types of cancer including ovarian, brain, stomach, and lung cancers.
- ERBB3 is also known as v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 is a member of the epidermal growth factor receptor family of receptor tyrosine kinases. Amplification of this gene and overexpression of the ERBB3 protein have been linked to numerous cancers, including prostate, bladder, and breast tumors. ERBB3 binds to and is activated by neuregulins and NTAK. Mutations and defects in the ERBB3 gene are the cause of the lethal congenital contracture syndrome type 2, also referred to as Israeli Bedouin multiple contracture syndrome type A. Israeli Bedouin multiple contracture syndrome type A is characterized by multiple joint contractures, anterior horn atrophy in the spinal chord, and a distended bladder.
- ERBB4 is also known as v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 is related to ERBB2 and ERBB3 and serves a similar function.
- Notch 1 is a member of the Type 1 transmembrane protein family and shares structural characteristics including an extracellular domain consisting of multiple epidermal growth factor-like repeats (EGF) and an intracellular domain consisting of multiple, different domain types.
- EGF epidermal growth factor-like repeats
- Notch proteins play a role in a variety of developmental processes by affecting the cell fate decisions.
- Notch genes have a signaling network that is an intracellular pathway which regulates interaction between physically adjacent cells. Defects in Notch 1 are a cause of bicuspid aortic valve, a common defect in the aortic valve where three heart leaflets are present instead of two. In rare cases, mutations in this gene can lead to restricted blood flow resulting in hypoplastic left heart syndrome. Aortic valve disease and T-cell acute lymphoblastic leukemia are associated with mutations in the Notch 1 gene.
- Notch 2 is related to Notch 1, as both proteins are members of the Notch family of receptor proteins.
- Notch 2 has five ligands: Jagged 1, Jagged 2, Delta-like 1, Delta-like 3, and Delta-like 4.
- the Notch 2 protein and its ligands send signals that are important prior to birth, with research indicating that signals triggered by the interaction between Notch 2 and its ligands contribute to the development of cells destined to be a part of the heart, liver, kidney, teeth, and other structures in the growing embryo.
- Notch 2 is involved in tissue repair after birth.
- Mutations in Notch 2 have been associated with Alagille syndrome. It is hypothesized that the mutations in Notch 2 probably result in a protein that is abnormally small or folded into an incorrect three-dimensional shape. Disrupted signaling in the Notc h2 gene is believed to cause development issues in the heart, liver, kidney and other parts of the body, resulting in the signs and symptoms of Alagille syndrome. Mutations in Notch 2 may be somatic mutations. These somatic mutations may lead to extra copies of the mutated gene, increasing gene activity. This increase in gene activity may lead to uncontrolled cell growth and cell division in the immune system cells, causing tumors.
- Notch 3 receptor protein is located on the surface of muscle cells that surround blood vessels. Notch 3 receptor protein is specific to the arteries; however, the protein is not present in veins. Signals are sent by Notch 3 to the nucleus of the cell such that particular genes are activated within vascular smooth muscle cells. The Notch 3 receptors are though to be essential for the maintenance of healthy muscle cells in the brain's arteries.
- Notch 3 is thought to be responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) due to one of 150 known mutations in the gene. Almost all of the 150 mutations change a single amino acid in the Notch 3 receptor. Disruption of the function of Notch 3 may lead to apoptosis of cells and damage to vascular smooth muscle cells. This damage to the vascular smooth muscle tissue is thought to cause recurrent strokes and other symptoms of CADASIL.
- CADASIL cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
- Notch 4 is related to Notch 1, Notch 2, and Notch 3 and serves a similar function.
- AKT1 or v-akt murine thymoma viral oncogene homolog 1
- AKT1 and related AKT2 are activated by platelet-derived growth factor. Growth factor-induced neuronal survival is mediated by AKTs. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates the components of the apoptosis machinery.
- AKT1 Mutations in AKT1 are associated with colorectal cancer and an increased susceptibility to ovarian cancer and familial breast-ovarian cancer type 1. Additionally, mutations in AKT1 have been linked to the susceptibility of schizophrenia.
- AKT2 or v-akt murine thymona viral oncogene homolog 2 belongs to the subfamily of serine threonine kinases containing SH2-like domains.
- AKT2 was shown to be amplified and overexpressed in ovarian cancer cell lines and primary ovarian tumors. The overexpression of the AKT2 protein contributes to a malignant phenotype of human ductal pancreatic cancers. The AKT2 gene and protein is indicated in diabetes mellitus.
- AKT3, or v-akt murine thymona viral oncogene homolog 3 encodes the AKT3 protein.
- the AKT3 gene is related to the AKT1 and AKT2 genes and mutations in the AKT3 gene caused overexpression of the AKT3 protein.
- HIF1A hypoxia-inducible factor 1, alpha subunit
- HIF1B hypoxia-inducible factor 1, beta subunit
- HIF1A is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau tumor suppressor protein leads to rapid degradation of the HIF1A protein.
- Polymorphisms in the HIF1A are associated with renal cell carcinoma phenotype. The use of immunohistochemical assessment of HIF1A can be used as a predictor of poor outcome may improve clinical decision making regarding adjuvant treatment of patients with lymph node negative breast carcinoma.
- Met met pronto-oncogene (hepatocyte growth factor receptor) is a hepatocyte growth factor receptor that has tyrosine-kinase activity.
- the activation of MET after rearrangement with the TPR gene produces an oncogenic protein.
- MET has also been associated with gastric cancer, hepatocellular carcinoma, hereditary papillary renal carcinoma, and other related conditions. Genetic variations and mutations in MET may also be associated with susceptibility to autism type 9.
- HRG or histidine-rich glycoprotein, contains two cysteine-like domains and is located in plasma and platelets. HRG can inhibit rosette formation and interacts with heparin, thrombospondin and plasminogen. There is a potential prothrombotic effect as exhibited by HRG's inhibition of fibrinolysis and the reduction of inhibition of coagulation. Mutations in HRG are thought to be the cause of thrombophilia due to histidine-rich glycoprotein deficiency.
- Bc12 is a tumor suppressor protein related to p53. As a tumor suppressor, Bc12 is involved in the replication of cells. Mutations in Bc12 can lead to uncontrolled or abnormal cell replication.
- Ppar(alpha), or peroxisome proliferator-activated receptor alpha is a nuclear receptor protein.
- Peroxisome proliferators include hypolipidemic drugs, herbicides, leukotriene antagonists, and plasticizers.
- Peroxisomes are subcellular organelles found in plants and animals that contain enzymes for respiration and for cholesterol and lipid metabolism. The action of peroxisomes is thought to be mediated via specific receptors, called PPARs, which affect the expression of target genes involved in cell proliferation, cell differentiation and in immune and inflammatory responses. Mutations in Ppar(alpha) have been linked to the susceptibility to hyperapobetalipoproteinemia.
- Ppar(gamma) or peroxisome proliferator-activated receptor gamma
- Ppar(gamma) regulates adipocyte differentiation that has been implicated in the pathology of numerous diseases such as obesity, diabetes, atherosclerosis, familial partial lipodystrophy type 3, and cancer. Mutations in Ppar(gamma) are associated with carotid intimal medial thickness 1. Other genetic variations in PPARG can be associated with susceptibility to glioma type 1.
- Gliomas are central nervous system neoplasms derived from glial cells and comprise astrocytomas, glioblastoma, multiforme, oligodendrogliomas, and ependymomas.
- WT1 is a transcription factor that regulates the activity of genes involved in cell growth and apoptosis.
- Wilmus tumor is a rare form of kidney cancer, where the individual has a mutation in one copy of the WT1 gene in every cell. In other types of Wilmus tumor, WT1 gene mutations are only present in tumor cells. The mutations in the WT1 genes are somatic, meaning they are not inherited. Mutations in the WT1 gene have also been linked to several other forms of cancer including lung, prostate, breast, and ovarian cancer, as well as leukemia, such as acute lymphoblastic leukemia, chronic myeloid leukemia, and childhood acute myeloid leukemia.
- Denys-Drash syndrome is also caused by a mutation in the WT1 gene.
- WT1 mutations are also thought to cause Frasier syndrome. The mutations disrupt the way the WT1 gene's instructions are used to make the protein resulting in a shortage of functional protein.
- the FGF receptor family or fibroblast growth factor receptor family includes FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.
- FGF receptors are involved in important processes such as cell division, regulation of cell growth and maturation, formation of blood vessels, wound healing, and embryonic development. Growth factors work with the FGF receptor proteins to signal chemical reactions within the cell that instruct the cell to undergo certain changes, such as maturing to take on special functions.
- the FGFR1 protein is thought to play a role in the development of the nervous system. Mutations in the FGFR1 gene are responsible for Kallmann syndrome, Pfeiffer syndrome, osteoglophonic dysplasia, and cancers such as pancreatic, esophageal, ovarian, testicular, breast, and head and neck cancers. Mutations in the FGFR2 gene have been lined to Apert syndrome, Beare-Stevenson cutis gyrate syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, lacrimo-auriculo-dento-digital (LADD) syndrome, and cancers such as prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, and head and neck cancers.
- Apert syndrome Beare-Stevenson cutis gyrate syndrome
- Crouzon syndrome Crouzon syndrome
- Jackson-Weiss syndrome Pfeiffer syndrome
- lacrimo-auriculo-dento-digital (LADD) syndrome and cancers such
- FGFR3 gene mutations have been associated with Achondroplasia, Crouzonodermoskeletal syndrome, hypochondroplasia, Muenke syndrome, SADDAN, thanatophoric dysplasia, bladder cancer, and platyspondylic lethal skeletal dysplasia.
- Several types of cancers are associated with mutations in the FGFR4 gene, such as breast, colon, gastric, pancreatic, ovarian, head and neck, and prostate. These cancers are usually linked to a polymorphism in which glycine is replaced by arginine at position 338 in the protein's chain of amino acids.
- CDKN2a or cyclin-dependent kinase inhibitor 2A, encodes the CDKN2a protein.
- the CDKN2a gene generates several transcript variants which differ in their first exons, with at least three alternatively spliced variants encoding distinct proteins, two of which encode structurally related isoforms known to function as inhibitors of CDK4 kinase. This interaction with CDK4 and with CDK6 allows CDKN2a to act as a negative regulator of the proliferation of normal cells. Further, CDKN2a is capable of inducing cell cycle arrest in G1 and G2 phases and acts as a tumor suppressor.
- CDKN2a Genetic variations in CDKN2a may underlie susceptibility to uveal melanoma, which is the most common type of ocular malignant tumor. Mutations in CDKN2a are also linked to cutaneous malignant melanoma type 2, familiar atypical multiple mole melanoma-pancreatic carcinoma syndrome, melanoma-astrocytoma syndrome, Li Fraumeni syndrome, melanoma and neural system tumor syndrome, orolaryngeal cancer, pancreatic cancer, and melanoma syndrome.
- APC is also referred to as adenomatous polyposis coli, plays a critical role in several cellular processes that determine whether a cell will develop into a tumor.
- the APC protein acts as a tumor suppressor, thus, it regulates the cell division cycle by keeping cells from growing and dividing too fast or in an uncontrolled way. Cell division, attachment and migration are all controlled, in part, by the APC protein.
- Mutations in the APC gene are associated with familial adenomatous polyposis, for which over 700 mutations have been identified. Cancers such as colorectal cancer, Turcot syndrome, colon cancer, and stomach cancer have been linked to mutations in the APC gene. Specifically, colon cancer has been linked to one mutation found in approximately 6% of people with Ashkenazi Jewish heritage that replaces isoleucine with lysine at position 1307 in the APC protein.
- Retinoblastoma 1, or Rb1 acts as a tumor suppressor, therefore, it regulates the cell cycle and prevents cells from dividing at a rapid pace or in an uncontrolled manner.
- the protein pRB can prevent other proteins from triggering DNA replication. Further, pRB interacts with other proteins to influence cell survival, apoptosis, and differentiation.
- Mutations in the RB1 gene have been associated with retinoblastoma, a rare type of eye cancer that typically affects young children. About 40% of retinoblastomas are germinal, and thus hereditary, while the other 60% of retinoblastomas are non-germinal, and thus, cannot be passed to the next generation.
- Bladder cancer, lung cancer, breast cancer, bone cancer, and melanoma have been associated with mutations in the RB1 gene.
- MEN1 (menin), also referred to multiple endocrine neoplasia I, and acts as a tumor suppressor and is likely involved in several important cell functions. Copying and repairing DNA and apoptosis are functions in which menin plays a role. Menin is also present in the nucleus of many different cell types and appears to be active in all stages of development. Menin also interacts with several transcription factors which bind to specific areas of DNA to help control the expression of the genes. Mutations in MEN1 have been linked to multiple endocrine neoplasia, wherein over 400 mutations in MEN1 have been identified.
- Familial isolated hyperparathyroidism has also been associated with genetic variation in MEN, as well as sporadic tumors, tumors of the parathyroid gland, pancreatic tumors, and cancerous tumors of the airways in the lungs called bronchial carcinoids.
- VHL or von-Hippel-Lindau tumor suppressor
- This complex targets other proteins to be degraded by the cell when they are no longer required. This degradation removes damaged or unnecessary proteins and helps maintain the normal functions of cells.
- a protein called hypoxia-inducible factor (HIF) is targeted by the VCB-CUL2 complex to be broken down within cells. HIF controls several important genes involved in cell division and the formation of new blood vessels. VHL protein has also been thought to play a role in other cellular functions, including the regulation of other genes and control of cell division, as well as the formation of extracellular matrix.
- VHL mutations in the VHL gene have been linked to von Hippel-Lindau syndrome, wherein over 370 inherited mutations have been identified. The mutations cause an altered or missing VHL protein, leading to a build up of HIF in the cell, signaling the cell to divide abnormally and trigger the production of unnecessary blood vessels. Kidney cancer has also been linked to somatic mutations in VHL. VHL mutations are also linked to hemangioblastoma, Chuvash polycythemia or congenital polycythemia.
- BRCA1 or breast cancer 1, early onset, is involved in repairing damaged DNA.
- BRCA1 protein interacts with several other proteins, including proteins produced by RAD51 and BARD1 genes, to mend breaks in DNA.
- BRCA1 plays a role in maintaining the stability of a cell's genetic information. It is thought that BRCA1 plays a critical role in embryonic development also. Mutations in BRCA1 are most strongly linked to breast cancer, wherein over 1,000 mutations in the BRCA1 gene have been identified. Most of these over 1,000 mutations lead to the production of an abnormally short BRCA1 protein. Other mutations in BRCA1 change single amino acids in the protein or delete large segments of DNA from the BRCA1 gene.
- BRCA1 The mutations in BRCA1 often result in a protein that is unable to adequately repair damaged DNA or fix mutations that occur in other genes, allowing cells to divide in an uncontrolled manner, forming a tumor. Mutations in BRCA1 are also associated with an increased risk of fallopian tube cancer, male breast cancer, and pancreatic cancer.
- BRCA2 or breast cancer 2, early onset, interacts with several other proteins, including RAD51 and BARD1, to mend breaks in DNA. By helping repair DNA, BRCA2 plays a role in maintaining the stability of a cell's genetic information. BRCA2 may also help regulate cytokinesis, a step in the cell division process where the cytoplasm divides to form two separate cells. BRCA2 is most strongly associated with breast cancer, wherein over 800 mutations in BRCA2 have been identified. These mutations insert or delete a small number of nucleotides in the gene, which disrupts protein production from one copy of the gene in each cell, resulting in an abnormally small, nonfunctional BRCA2 protein.
- BRCA2 mutations have also been associated with Fanconi anemia type D1, which results when two faulty copies of the BRCA2 gene are present in each cell.
- Fanconi anemia type D1 An increased risk of ovarian cancer, prostate cancer, pancreatic cancer, fallopian tube cancer, male breast cancer, and melanoma is associated with genetic variations in BRCA2.
- AR or androgen receptor
- Androgen receptors help direct the development of male sexual characteristics, as well as regulating hair growth and sex drive in females.
- Mutations in the AR gene have been known to cause androgen insensitivity syndrome. Most of the mutations leading to androgen insensitivity syndrome cause changes in single base pairs of DNA, sometimes leading to a shortened version of the AR protein or leading to an abnormal receptor that cannot bind to androgens or to DNA.
- Other disorders associated with genetic variation in the AR protein include spinal and bulbar muscular atrophy, androgenetic alopecia, breast cancer, prostate cancer, and endometrial cancer in women.
- TSG101 belongs to a group of inactive homologs of ubiquitin-conjugating enzymes.
- the protein may play a role in cell growth and differentiation and acts as a negative growth regulator.
- Genomic stability and cell cycle regulation appear to be linked to the in vivo steady-state expression of TSG101, which acts as a tumor suppressor. Mutations in the TSG101 gene occur in high frequency in breast cancer.
- Igf1 and Igf2, or insulin-like growth factor 1 and insulin-like growth factor 2 are structurally and functionally related to insulin but have a much higher growth-promoting activity.
- Igf2 is thought to play an essential role in growth and development before birth. With regard to the inheritance of Igf2, the copy of the gene inherited from a person's father is the only active copy in most parts of the body. Mutations in Igf1 have been linked to insulin-like growth factor 1 deficiency, an autosomal recessive disorder characterized by growth retardation, sensorineural deafness, and mental retardation.
- Igf2 Mutations in Igf2 are associated with Beckwith-Wiedmann syndrome and an increased susceptibility to cancers such as Wilmus tumor, heptaoblastoma, embryonal tumors, Russell-Silver syndrome, breast cancer, prostate cancer, lung cancer, colon cancer, and liver cancer. Normal variations in the Igf2 gene may also be involved in determining the adult height and/or weight of an individual.
- cancers such as Wilmus tumor, heptaoblastoma, embryonal tumors, Russell-Silver syndrome, breast cancer, prostate cancer, lung cancer, colon cancer, and liver cancer.
- Normal variations in the Igf2 gene may also be involved in determining the adult height and/or weight of an individual.
- Igf 1R insulin-like growth factor 1 receptor
- Igf 2R insulin-like growth factor 2 receptor
- Igf 1R plays a critical role in transformation events.
- Igf 2R is a receptor for both insulin-like growth factor 2 and mannose 6-phosphate. Genetic variance in Igf 1R has been associated with a growth deficiency disorder characterized by intrauterine growth retardation and poor postnatal growth accompanied by increased plasma Igf1. Mutations in Igf 2R are associated with heptocellular carcinoma.
- Bax or BCL-2 associated X protein, encodes for the BCL2 protein.
- BCL2 family member proteins form hetero-or homodimers and act as anti- or pro-apoptotic regulators that are involved in a variety of cellular activities.
- the expression of the Bax gene is regulated by the tumor suppressor p53 and has been shown to be involved in p53-mediated apoptosis. Mutations in the Bax gene have been associated with colorectal cancer and t-cell acute lymphoblastic leukemia.
- the caspase family CASP1, CASP2, CASP3, CASP4, CASP6, CASP7, CASP8, CASP9, and CASP12; are a family of proteases responsible for carrying out the cell death process.
- the proteases are kept active by proteins on the mitochondrial cell surface from the BCL2 family.
- BCL2 function is blocked and caspase activators initiate the cell death cascade.
- CASP8, caspase 8 is associated with autoimmune lymphoproliferative syndrome, hepatocellular carcinoma, somatic, and lung cancer.
- CASP12 is associated with the susceptibility to sepsis.
- Kras or v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog, is primarily involved in regulating cell division.
- K-Ras uses signal transduction to relay signals from the outside of the cell into the cell's nucleus. The signals provided by K-Ras instruct the cell to grow, divide, and differentiate.
- K-Ras is a GTPase, meaning that it converts GTP to GDP, thus, K-Ras acts like a switch that is turned on and off by the GTP and GDP molecules.
- the Kras gene belongs to a class of genes known as oncogenes that, when mutated, have the potential to cause normal cells to become cancerous. Mutations in Kras have been linked to Noonan Syndrome, several types of cancers, including pancreatic, lung, and colorectal cancers, cardiofaciocutaneous syndrome, and Costello syndrome.
- Pten acts as a tumor suppressor, thus, helps regulate the cycle of cell division by keeping cells from growing and dividing too rapidly or in an uncontrolled way.
- PTEN modifies other proteins and lipids by removing phosphate groups.
- the PTEN enzyme acts as part of a chemical pathway that signals the cell to stop dividing and triggers cells to undergo a form of programmed cell death, called apoptosis.
- PTEN acts to control cell growth so that it does not become irregular or uncontrolled, leading to cancer. Mutations in the PTEN gene have been associated with Cowden syndrome, breast cancer, Bannayan-Riley-Rubalcaba syndrome, Proteus syndrome, and Proteus-like syndrome. These disorders are collectively referred to as PTEN hamartoma tumor syndromes (PHTS).
- PHTS PTEN hamartoma tumor syndromes
- BCRP breast cancer resistance protein
- BCRP breast cancer resistance protein
- BRCP is also known as ABCG2.
- BRCP is an ATP-binding transport protein that is expressed in several organs, including the liver.
- BCRP is an ABC transport protein and these transport proteins have been shown to play an important pathophysiological role in several liver diseases.
- P53 is a nuclear localized phosphoprotein. P53 is thought to be involved in transcription regulation. Phosphorylation regulates the activity of p53 and the level of p53 is low after mitosis, but increases after G 1 . Further, p53 may also regulate the initiation of DNA synthesis. Due to the involvement of p53 in both transcription and DNA replication, the various mutants of p53 may also regulate the initiation of DNA synthesis.
- animal refers to a non-human animal.
- the animal may be an embryo, a juvenile, or an adult.
- Suitable animals include vertebrates such as mammals, birds, reptiles, amphibians, and fish. Examples of suitable mammals include without limit rodents, companion animals, livestock, and primates.
- rodents include mice, rats, hamsters, gerbils, and guinea pigs.
- Suitable companion animals include but are not limited to cats, dogs, rabbits, hedgehogs, and ferrets.
- livestock include horses, goats, sheep, swine, cattle, llamas, and alpacas.
- Suitable primates include, but are not limited to, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys.
- birds include chickens, turkeys, ducks, and geese.
- the animal may be an invertebrate such as an insect, a nematode, and the like.
- insects include Drosophila and mosquitoes.
- An exemplary animal is a rat.
- Non-limiting examples of commonly used rat strains suitable for genetic manipulation include Dahl Salt-Sensitive, Fischer 344, Lewis, Long Evans Hooded, Sprague-Dawley and Wistar.
- the animal does not comprise a genetically modified mouse.
- the animal does not include exogenously introduced, randomly integrated transposon sequences.
- the protein involved in tumor suppression may be endogenous to the animal or it may be exogenous (such as an orthologous tumor suppressor protein). Exogenous proteins may be from any of the animals listed above, as well as from human.
- the type of genetically modified animal and the source of the protein involved in tumor suppression can and will vary.
- the genetically modified animal may be a rat, cat, dog, or pig, and the protein involved in tumor suppression may be human.
- the animal is a rat and preferred sequences involved in tumor suppression are listed below.
- the genetically modified animal comprises at least one chromosomally integrated sequence encoding an exogenous protein involved in tumor suppression
- the exogenous protein is human.
- the genetically modified animal is a rat and the exogenous protein involved in tumor suppression is human.
- a further aspect of the present disclosure provides genetically modified cells or cell lines comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression.
- the genetically modified cell or cell line may be derived from any of the genetically modified animals disclosed herein.
- the chromosomal sequence involved in tumor suppression may be edited in a cell as detailed below.
- the disclosure also encompasses a lysate of said cells or cell lines.
- the cells will be eukaryotic cells.
- Suitable host cells include fungi or yeast, such as Pichia, Saccharomyces, or Schizosaccharomyces; insect cells, such as SF9 cells from Spodoptera frugiperda or S2 cells from Drosophila melanogaster; and animal cells, such as mouse, rat, hamster, non-human primate, or human cells.
- Exemplary cells are mammalian.
- the mammalian cells may be primary cells. In general, any primary cell that is sensitive to double strand breaks may be used.
- the cells may be of a variety of cell types, e.g., fibroblast, myoblast, T or B cell, macrophage, epithelial cell, and so forth.
- the cell line may be any established cell line or a primary cell line that is not yet described.
- the cell line may be adherent or non-adherent, or the cell line may be grown under conditions that encourage adherent, non-adherent or organotypic growth using standard techniques known to individuals skilled in the art.
- Non-limiting examples of suitable mammalian cell lines include Chinese hamster ovary (CHO) cells, monkey kidney CVI line transformed by SV40 (COS7), human embryonic kidney line 293, baby hamster kidney cells (BHK), mouse sertoli cells (TM4), monkey kidney cells (CVI-76), African green monkey kidney cells (VERO), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT), rat hepatoma cells (HTC), HIH/3T3 cells, the human U2-OS osteosarcoma cell line, the human A549 cell line, the human K562 cell line, the human HEK293 cell lines, the human HEK293T cell line, and TRI cells.
- ATCC® American Type Culture Collection catalog
- the cell may be a stem cell.
- Suitable stem cells include without limit embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, and unipotent stem cells.
- the genetically modified animal or cell detailed above in sections (I) and (II), respectively, is generated using a zinc finger nuclease-mediated genome editing process.
- the process for editing a chromosomal sequence comprises: (a) introducing into an embryo or cell at least one nucleic acid encoding a zinc finger nuclease that recognizes a target sequence in the chromosomal sequence and is able to cleave a site in the chromosomal sequence, and, optionally, (i) at least one donor polynucleotide comprising a sequence for integration flanked by an upstream sequence and a downstream sequence that share substantial sequence identity with either side of the cleavage site, or (ii) at least one exchange polynucleotide comprising a sequence that is substantially identical to a portion of the chromosomal sequence at the cleavage site and which further comprises at least one nucleotide change; and (b) culturing the embryo or cell to allow expression of the zinc finger nucle
- the method comprises, in part, introducing into an embryo or cell at least one nucleic acid encoding a zinc finger nuclease.
- a zinc finger nuclease comprises a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease).
- the DNA binding and cleavage domains are described below.
- the nucleic acid encoding a zinc finger nuclease may comprise DNA or RNA.
- the nucleic acid encoding a zinc finger nuclease may comprise mRNA.
- the nucleic acid encoding a zinc finger nuclease comprises mRNA
- the mRNA molecule may be 5′ capped.
- the nucleic acid encoding a zinc finger nuclease comprises mRNA
- the mRNA molecule may be polyadenylated.
- An exemplary nucleic acid according to the method is a capped and polyadenylated mRNA molecule encoding a zinc finger nuclease. Methods for capping and polyadenylating mRNA are known in the art.
- Zinc finger binding domains may be engineered to recognize and bind to any nucleic acid sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; and Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416.
- An engineered zinc finger binding domain may have a novel binding specificity compared to a naturally-occurring zinc finger protein.
- Rational design includes, for example, using databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence.
- databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence.
- U.S. Pat. Nos. 6,453,242 and 6,534,261 the disclosures of which are incorporated by reference herein in their entireties.
- the algorithm of described in U.S. Pat. No. 6,453,242 may be used to design a zinc finger binding domain to target a preselected sequence.
- Alternative methods such as rational design
- a zinc finger binding domain may be designed to recognize a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, or from about 8 to about 19 nucleotides in length.
- the zinc finger binding domains of the zinc finger nucleases disclosed herein comprise at least three zinc finger recognition regions (i.e., zinc fingers).
- the zinc finger binding domain may comprise four zinc finger recognition regions.
- the zinc finger binding domain may comprise five zinc finger recognition regions.
- the zinc finger binding domain may comprise six zinc finger recognition regions.
- a zinc finger binding domain may be designed to bind to any suitable target DNA sequence. See for example, U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, the disclosures of which are incorporated by reference herein in their entireties.
- Exemplary methods of selecting a zinc finger recognition region may include phage display and two-hybrid systems, and are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237, each of which is incorporated by reference herein in its entirety.
- enhancement of binding specificity for zinc finger binding domains has been described, for example, in WO 02/077227.
- Zinc finger binding domains and methods for design and construction of fusion proteins are known to those of skill in the art and are described in detail in U.S. Patent Application Publication Nos. 20050064474 and 20060188987, each incorporated by reference herein in its entirety.
- Zinc finger recognition regions and/or multi-fingered zinc finger proteins may be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length. See, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, the disclosures of which are incorporated by reference herein in their entireties, for non-limiting examples of linker sequences of six or more amino acids in length.
- the zinc finger binding domain described herein may include a combination of suitable linkers between the individual zinc fingers of the protein.
- the zinc finger nuclease may further comprise a nuclear localization signal or sequence (NLS).
- NLS nuclear localization signal or sequence
- a NLS is an amino acid sequence which facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome.
- Nuclear localization signals are known in the art. See, for example, Makkerh et al. (1996) Current Biology 6:1025-1027.
- An exemplary zinc finger DNA binding domain recognizes and binds a sequence having at least about 80% sequence identity with a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8.
- the sequence identity may be about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
- a zinc finger nuclease also includes a cleavage domain.
- the cleavage domain portion of the zinc finger nucleases disclosed herein may be obtained from any endonuclease or exonuclease.
- Non-limiting examples of endonucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388 or www.neb.com.
- cleave DNA e.g., S1 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease. See also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains.
- a cleavage domain also may be derived from an enzyme or portion thereof, as described above, that requires dimerization for cleavage activity.
- Two zinc finger nucleases may be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer.
- a single zinc finger nuclease may comprise both monomers to create an active enzyme dimer.
- an “active enzyme dimer” is an enzyme dimer capable of cleaving a nucleic acid molecule.
- the two cleavage monomers may be derived from the same endonuclease (or functional fragments thereof), or each monomer may be derived from a different endonuclease (or functional fragments thereof).
- the recognition sites for the two zinc finger nucleases are preferably disposed such that binding of the two zinc finger nucleases to their respective recognition sites places the cleavage monomers in a spatial orientation to each other that allows the cleavage monomers to form an active enzyme dimer, e.g., by dimerizing.
- the near edges of the recognition sites may be separated by about 5 to about 18 nucleotides. For instance, the near edges may be separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides.
- any integral number of nucleotides or nucleotide pairs may intervene between two recognition sites (e.g., from about 2 to about 50 nucleotide pairs or more).
- the near edges of the recognition sites of the zinc finger nucleases such as for example those described in detail herein, may be separated by 6 nucleotides.
- the site of cleavage lies between the recognition sites.
- Restriction endonucleases are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding.
- Certain restriction enzymes e.g., Type IIS
- Fok I catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al.
- a zinc finger nuclease may comprise the cleavage domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
- Type IIS restriction enzymes are described for example in International Publication WO 07/014,275, the disclosure of which is incorporated by reference herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.
- Fok I An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I.
- This particular enzyme is active as a dimmer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575).
- the portion of the Fok I enzyme used in a zinc finger nuclease is considered a cleavage monomer.
- two zinc finger nucleases, each comprising a Fok I cleavage monomer may be used to reconstitute an active enzyme dimer.
- a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage monomers may also be used.
- the cleavage domain may comprise one or more engineered cleavage monomers that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 20050064474, 20060188987, and 20080131962, each of which is incorporated by reference herein in its entirety.
- amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets for influencing dimerization of the Fok I cleavage half-domains.
- Exemplary engineered cleavage monomers of Fok I that form obligate heterodimers include a pair in which a first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of Fok I and a second cleavage monomer that includes mutations at amino-acid residue positions 486 and 499.
- a mutation at amino acid position 490 replaces Glu (E) with Lys (K); a mutation at amino acid residue 538 replaces Iso (I) with Lys (K); a mutation at amino acid residue 486 replaces Gln (Q) with Glu (E); and a mutation at position 499 replaces Iso (I) with Lys (K).
- the engineered cleavage monomers may be prepared by mutating positions 490 from E to K and 538 from I to K in one cleavage monomer to produce an engineered cleavage monomer designated “E490K:1538K” and by mutating positions 486 from Q to E and 499 from I to L in another cleavage monomer to produce an engineered cleavage monomer designated “Q486E:I499L.”
- the above described engineered cleavage monomers are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished.
- Engineered cleavage monomers may be prepared using a suitable method, for example, by site-directed mutagenesis of wild-type cleavage monomers (Fok I) as described in U.S. Patent Publication No. 20050064474 (see Example 5).
- the zinc finger nuclease described above may be engineered to introduce a double stranded break at the targeted site of integration.
- the double stranded break may be at the targeted site of integration, or it may be up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 1000 nucleotides away from the site of integration.
- the double stranded break may be up to 1, 2, 3, 4, 5, 10, 15, or 20 nucleotides away from the site of integration.
- the double stranded break may be up to 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides away from the site of integration.
- the double stranded break may be up to 50, 100, or 1000 nucleotides away from the site of integration.
- the method for editing chromosomal sequences involved in tumor suppression may further comprise introducing at least one donor polynucleotide comprising a sequence encoding a protein involved in tumor suppression into the embryo or cell.
- a donor polynucleotide comprises at least three components: the sequence coding the protein involved in tumor suppression, an upstream sequence, and a downstream sequence.
- the sequence encoding the protein involved in tumor suppression is flanked by the upstream and downstream sequence, wherein the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome.
- the donor polynucleotide will be DNA.
- the donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
- An exemplary donor polynucleotide comprising the sequence encoding a protein involved in tumor suppression may be a BAC.
- the sequence of the donor polynucleotide that encodes the protein involved in tumor suppression may include coding (i.e., exon) sequence, as well as intron sequences and upstream regulatory sequences (such as, e.g., a promoter).
- coding i.e., exon
- intron sequences e.g., a promoter
- upstream regulatory sequences such as, e.g., a promoter
- the size of the sequence encoding the protein involved in tumor suppression can and will vary.
- the sequence encoding the protein involved in tumor suppression may range in size from about 1 kb to about 5,000 kb.
- the donor polynucleotide also comprises upstream and downstream sequences flanking the chromosomal sequence involved in tumor suppression.
- the upstream and downstream sequences in the donor polynucleotide are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide.
- the upstream sequence refers to a nucleic acid sequence that shares sequence similarity with the chromosomal sequence upstream of the targeted site of integration.
- the downstream sequence refers to a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration.
- the upstream and downstream sequences in the donor polynucleotide may share about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted chromosomal sequence. In other embodiments, the upstream and downstream sequences in the donor polynucleotide may share about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted chromosomal sequence. In an exemplary embodiment, the upstream and downstream sequences in the donor polynucleotide may share about 99% or 100% sequence identity with the targeted chromosomal sequence.
- An upstream or downstream sequence may comprise from about 50 bp to about 2500 bp.
- an upstream or downstream sequence may comprise about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
- An exemplary upstream or downstream sequence may comprise about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp.
- the donor polynucleotide may further comprise a marker.
- a marker may make it easy to screen for targeted integrations.
- suitable markers include restriction sites, fluorescent proteins, or selectable markers.
- a double stranded break introduced into the chromosomal sequence by the zinc finger nuclease is repaired, via homologous recombination with the donor polynucleotide, such that the chromosomal sequence involved in tumor suppression is integrated into the chromosome.
- the presence of a double-stranded break facilitates integration of the sequence into the chromosome.
- a donor polynucleotide may be physically integrated or, alternatively, the donor polynucleotide may be used as a template for repair of the break, resulting in the introduction of the chromosomal sequence involved in tumor suppression as well as all or part of the upstream and downstream sequences of the donor polynucleotide into the chromosome.
- endogenous chromosomal sequence may be converted to the sequence of the donor polynucleotide.
- the method for editing chromosomal sequences involved in tumor suppression may further comprise introducing into the embryo or cell at least one exchange polynucleotide comprising a sequence that is substantially identical to the chromosomal sequence at the site of cleavage and which further comprises at least one specific nucleotide change.
- the exchange polynucleotide will be DNA.
- the exchange polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
- An exemplary exchange polynucleotide may be a DNA plasmid.
- the sequence in the exchange polynucleotide is substantially identical to a portion of the chromosomal sequence at the site of cleavage.
- the sequence of the exchange polynucleotide will share enough sequence identity with the chromosomal sequence such that the two sequences may be exchanged by homologous recombination.
- the sequence in the exchange polynucleotide may have at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with a portion of the chromosomal sequence.
- the sequence in the exchange polynucleotide comprises at least one specific nucleotide change with respect to the sequence of the corresponding chromosomal sequence.
- one nucleotide in a specific codon may be changed to another nucleotide such that the codon codes for a different amino acid.
- the sequence in the exchange polynucleotide may comprise one specific nucleotide change such that the encoded protein comprises one amino acid change.
- the sequence in the exchange polynucleotide may comprise two, three, four, or more specific nucleotide changes such that the encoded protein comprises one, two, three, four, or more amino acid changes.
- sequence in the exchange polynucleotide may comprise a three nucleotide deletion or insertion such that the reading frame of the coding reading is not altered (and a functional protein is produced).
- the expressed protein would comprise a single amino acid deletion or insertion.
- the length of the sequence in the exchange polynucleotide that is substantially identical to a portion of the chromosomal sequence at the site of cleavage can and will vary.
- the sequence in the exchange polynucleotide may range from about 50 bp to about 10,000 bp in length.
- the sequence in the exchange polynucleotide may be about 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 bp in length.
- the sequence in the exchange polynucleotide may be about 5500, 6000, 6500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bp in length.
- a double stranded break introduced into the chromosomal sequence by the zinc finger nuclease is repaired, via homologous recombination with the exchange polynucleotide, such that the sequence in the exchange polynucleotide may be exchanged with a portion of the chromosomal sequence.
- the presence of the double stranded break facilitates homologous recombination and repair of the break.
- the exchange polynucleotide may be physically integrated or, alternatively, the exchange polynucleotide may be used as a template for repair of the break, resulting in the exchange of the sequence information in the exchange polynucleotide with the sequence information in that portion of the chromosomal sequence.
- a portion of the endogenous chromosomal sequence may be converted to the sequence of the exchange polynucleotide.
- the changed nucleotide(s) may be at or near the site of cleavage. Alternatively, the changed nucleotide(s) may be anywhere in the exchanged sequences. As a consequence of the exchange, however, the chromosomal sequence is modified.
- At least one nucleic acid molecule encoding a zinc finger nuclease and, optionally, at least one exchange polynucleotide or at least one donor polynucleotide are delivered to the embryo or the cell of interest.
- the embryo is a fertilized one-cell stage embryo of the species of interest.
- Suitable methods of introducing the nucleic acids to the embryo or cell include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.
- the nucleic acids may be introduced into an embryo by microinjection.
- the nucleic acids may be microinjected into the nucleus or the cytoplasm of the embryo.
- the nucleic acids may be introduced into a cell by nucleofection.
- the ratio of donor (or exchange) polynucleotide to nucleic acid encoding a zinc finger nuclease may range from about 1:10 to about 10:1.
- the ratio of donor (or exchange) polynucleotide to nucleic acid encoding a zinc finger nuclease may be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In one embodiment, the ratio may be about 1:1.
- nucleic acids may be introduced simultaneously or sequentially.
- nucleic acids encoding the zinc finger nucleases, each specific for a distinct recognition sequence, as well as the optional donor (or exchange) polynucleotides may be introduced at the same time.
- each nucleic acid encoding a zinc finger nuclease, as well as the optional donor (or exchange) polynucleotides may be introduced sequentially.
- the embryo may be derived from an animal comprising at least one edited chromosomal sequence involved in tumor suppression.
- an animal comprising 2, 3, 4, or more edited chromosomal sequences involved in tumor suppression may be generated.
- the method of inducing genomic editing with a zinc finger nuclease further comprises culturing the embryo or cell comprising the introduced nucleic acid(s) to allow expression of the zinc finger nuclease.
- An embryo may be cultured in vitro (e.g., in cell culture). Typically, the embryo is cultured at an appropriate temperature and in appropriate media with the necessary O 2 /CO 2 ratio to allow the expression of the zinc finger nuclease. Suitable non-limiting examples of media include M2, M16, KSOM, BMOC, and HTF media.
- M2 M16
- KSOM KSOM
- BMOC BMOC
- HTF media a cell line may be derived from an in vitro-cultured embryo (e.g., an embryonic stem cell line).
- an embryo may be cultured in vivo by transferring the embryo into the uterus of a female host.
- the female host is from the same or similar species as the embryo.
- the female host is pseudo-pregnant.
- Methods of preparing pseudo-pregnant female hosts are known in the art.
- methods of transferring an embryo into a female host are known. Culturing an embryo in vivo permits the embryo to develop and may result in a live birth of an animal derived from the embryo. Such an animal would comprise the edited chromosomal sequence involved in tumor suppression in every cell of the body.
- cells comprising the introduced nucleic acids may be cultured using standard procedures to allow expression of the zinc finger nuclease.
- Standard cell culture techniques are described, for example, in Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651; and Lombardo et al (2007) Nat. Biotechnology 25:1298-1306.
- Routine optimization may be used, in all cases, to determine the best techniques for a particular cell type.
- the chromosomal sequence may be edited.
- the zinc finger nuclease recognizes, binds, and cleaves the target sequence in the chromosomal sequence of interest.
- the double-stranded break introduced by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process. Consequently, a deletion, insertion, or point mutation may be introduced in the chromosomal sequence such that the sequence is inactivated.
- the zinc finger nuclease recognizes, binds, and cleaves the target sequence in the chromosome.
- the double-stranded break introduced by the zinc finger nuclease is repaired, via homologous recombination with the donor (or exchange) polynucleotide, such that the sequence in the donor polynucleotide is integrated into the chromosomal sequence (or a portion of the chromosomal sequence is converted to the sequence in the exchange polynucleotide).
- a sequence may be integrated into the chromosomal sequence (or a portion of the chromosomal sequence may be modified).
- the genetically modified animals disclosed herein may be crossbred to create animals comprising more than one edited chromosomal sequence or to create animals that are homozygous for one or more edited chromosomal sequences.
- two animals comprising the same edited chromosomal sequence may be crossbred to create an animal homozygous for the edited chromosomal sequence.
- animals with different edited chromosomal sequences may be crossbred to create an animal comprising both edited chromosomal sequences.
- animal A comprising an inactivated ATM chromosomal sequence may be crossed with animal B comprising a chromosomally integrated sequence encoding a human ATM protein to give rise to a “humanized” ATM 1 offspring comprising both the inactivated ATM chromosomal sequence and the chromosomally integrated human ATM sequence.
- animal B comprising a chromosomally integrated sequence encoding a human ATM protein may be crossed with animal comprising a chromosomally integrated sequence encoding the human Notch 1 protein to generate “humanized” Notch 1 offspring.
- a humanized ATM animal may be crossed with a humanized Notch 1 animal to create a humanized ATM/Notch 1 animal.
- an animal comprising an edited chromosomal sequence disclosed herein may be crossbred to combine the edited chromosomal sequence with other genetic backgrounds.
- other genetic backgrounds may include wild-type genetic backgrounds, genetic backgrounds with deletion mutations, genetic backgrounds with other targeted integrations, and genetic backgrounds with non-targeted integrations.
- a further aspect of the present disclosure encompasses methods for using the genetically modified animals.
- a genetically modified animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to determine susceptibility to developing tumors.
- the method comprises exposing the genetically modified animal comprising an inactivated tumor suppressor sequence and a wild-type animal to a carcinogenic agent, and then monitoring the development of tumors.
- the animal comprising the inactivated tumor suppressor sequence may have an increased risk for tumor formation.
- an animal homozygous for the inactivated tumor suppressor sequence may have increased risk relative to an animal heterozygous for the same inactivated sequence, which in turn may have increased risk relative to a wild-type animal.
- a similar method may be used to screen for spontaneous tumors, wherein the animals are not exposed to a carcinogenic agent.
- an animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to evaluate the carcinogenic potential of a test agent.
- the method comprises contacting the genetically modified animal comprising an inactivated tumor suppressor sequence and a wild-type animal to the test agent, and then monitoring the development of tumors. If the animal comprising an inactivated tumor suppressor sequence has an increased incidence of tumors relative to the wild-type animal, the test agent may be carcinogenic.
- an animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to determine the efficacy and/or toxicity of a chemotherapeutic agent or a combination of chemotherapeutic agents.
- the method comprises inducing tumor formation in genetically modified animal comprising inactivated tumor suppressor sequences, and then comparing the responses of a first group of animals contacted with the chemotherapeutic agent or combination of chemotherapeutic agents to a second group of animal not contacted with the chemotherapeutic agent or combination of chemotherapeutic agents.
- an animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to screen libraries of small molecule drugs for potentially advantageous effects, including enhanced potency as well as reduced untoward effects.
- the method comprises inducing tumor formation in genetically modified animal comprising inactivated tumor suppressor sequences, and then comparing the responses of a first group of animals contacted with the small molecule drug candidate to a second group of animal not contacted with the small molecule drug candidate.
- a genetically modified animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used test the ADME/Tox profile of a chemotherapeutic agent or a combination of chemotherapeutic agents.
- assessment parameters include damage to DNA, metabolic consequence, and behavioral effects of the chemotherapeutic agent or the combination of chemotherapeutic agents.
- Behavioral tests include test of learning/memory, anxiety/depression, and sensori-motor functions.
- Non-limiting examples of behavioral tests suitable for assessing the motor function of rats includes open field locomoter activity assessment, the rotarod test, the grip strength test, the cylinder test, the limb-placement or grid walk test, the vertical pole test, the Inverted grid test, the adhesive removal test, the painted paw or catwalk (gait) tests, the beam traversal test, and the inclined plane test.
- Non-limiting examples of behavioral tests suitable for assessing the long-term memory function of rats include the elevated plus maze test, the Morris water maze swim test, contextual fear conditioning, the Y-maze test, the T-maze test, the novel object recognition test, the active avoidance test, the passive (inhibitory) avoidance test, the radial arm maze test, the two-choice swim test, the hole board test, the olfactory discrimination (go-no-go) test, and the pre-pulse inhibition test.
- Non-limiting examples of behavioral tests suitable for assessing the anxiety of rats include the open field locomotion assessment, observations of marble-burying behavior, the elevated plus maze test, the light/dark box test.
- Non-limiting examples of behavioral tests suitable for assessing the depression of rats includes the forced swim test, the tail suspension test, the hot plate test, the tail suspension test, anhedonia observations, and the novelty suppressed feeding test.
- the genetically modified animals disclosed herein may be used for gene therapy.
- an animal having a natural mutation in a tumor suppressor gene may genetically modified by editing the chromosomal sequence comprising the natural mutation such that the mutation is corrected. Accordingly, the animal may no longer be susceptible to tumor formation or cancer development.
- Still yet another aspect encompasses a method of generating a cell line or cell lysate using a genetically modified animal comprising an edited chromosomal sequence involved in tumor suppression.
- An additional other aspect encompasses a method of producing purified biological components using a genetically modified cell or animal comprising an edited chromosomal sequence involved in tumor suppression.
- biological components include antibodies, receptor proteins, altered tumor suppressor proteins, and the like.
- chromosomal sequence involved in tumor suppression refers to a chromosomal sequence which has been identified to contribute to cell cycle maintenance, division of cells, and/or the cell death cycle. Any chromosomal sequence thought to be involved in tumor suppression will work for purposes of the present invention.
- Exemplary chromosomal sequences involved in tumor suppression include, but are not limited to, ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor
- a protein encoded by a chromosomal sequence involved in tumor suppression or “a protein involved in tumor suppression” refers to a protein that has been encoded by a chromosomal sequence which has been identified to contribute to cell cycle maintenance, division of cells, and/or the cell death cycle. Any chromosomal sequence thought to be involved in tumor suppression will work for purposes of the present invention.
- Exemplary chromosomal sequences involved in tumor suppression include, but are not limited to, ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor
- a “gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.
- nucleic acid and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer.
- the terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T.
- polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues.
- recombination refers to a process of exchange of genetic information between two polynucleotides.
- homologous recombination refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells. This process requires sequence similarity between the two polynucleotides, uses a “donor” or “exchange” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
- such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
- Such specialized homologous recombination often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
- target site or “target sequence” refer to a nucleic acid sequence that defines a portion of a chromosomal sequence to be edited and to which a zinc finger nuclease is engineered to recognize and bind, provided sufficient conditions for binding exist.
- nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity.
- the percent identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
- An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
- the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between regions that share a degree of sequence identity, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
- Two nucleic acid, or two polypeptide sequences are substantially similar to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more-preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above.
- substantially similar also refers to sequences showing complete identity to a specified DNA or polypeptide sequence.
- DNA sequences that are substantially similar can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
- Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
- hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
- Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
- a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
- a nucleic acid probe When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a reference nucleic acid sequence, and then by selection of appropriate conditions the probe and the reference sequence selectively hybridize, or bind, to each other to form a duplex molecule.
- a nucleic acid molecule that is capable of hybridizing selectively to a reference sequence under moderately stringent hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
- Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
- Hybridization conditions useful for probe/reference sequence hybridization where the probe and reference sequence have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press). Conditions for hybridization are well-known to those of skill in the art.
- Hybridization stringency refers to the degree to which hybridization conditions disfavor the formation of hybrids containing mismatched nucleotides, with higher stringency correlated with a lower tolerance for mismatched hybrids.
- Factors that affect the stringency of hybridization include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as, for example, formamide and dimethylsulfoxide.
- hybridization stringency is increased by higher temperatures, lower ionic strength and lower solvent concentrations.
- stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of the sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
- a particular set of hybridization conditions may be selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
- the p53 gene was chosen for zinc finger nuclease (ZFN) mediated genome editing. ZFNs were designed, assembled, and validated using strategies and procedures previously described (see Geurts et al. Science (2009) 325:433). ZFN design made use of an archive of pre-validated 1-finger and 2-finger modules.
- the rat p53 gene region (NM — 030989) was scanned for putative zinc finger binding sites to which existing modules could be fused to generate a pair of 4-, 5-, or 6-finger proteins that would bind a 12-18 bp sequence on one strand and a 12-18 bp sequence on the other strand, with about 5-6 bp between the two binding sites.
- mRNA encoding each pair of ZFNs was produced using known molecular biology techniques.
- the mRNA was transfected into rat cells.
- Control cells were transfected with mRNA encoding GFP.
- Active ZFN pairs were identified by detecting ZFN-induced double strand chromosomal breaks using the Cel-1 nuclease assay. This assay detects alleles of the target locus that deviate from wild type as a result of non-homologous end joining (NHEJ)-mediated imperfect repair of ZFN-induced DNA double strand breaks.
- NHEJ non-homologous end joining
- polyadenylated mRNA encoding the active pair of ZFNs was microinjected into fertilized rat embryos using standard procedures (e.g., see Geurts et al. (2009) supra). Control embryos were microinjected with saline or mRNA encoding GFP. The injected embryos were transferred to pseudopregnant female rats to be carried to parturition. Toe/tail of clips of each live born animal was harvested for DNA extraction and analysis using a Cel-1 assay. As shown in FIG. 1 , about 25% of the experimental animals had an edited p53 gene locus.
- ZFNs that target and cleave the BCRP gene were identified essentially as described above in Example 1.
- the rat BCRP gene (NM — 1811381) was scanned for putative zinc finger binding sites.
- ZFNs were assembled and tested essentially as described in Example 1. It was found that the ZFN pair targeted to bind 5′-atGACGTCAAGGAAGAAgtctgcagggt-3′ (SEQ ID NO:5) and 5′-acGGAGATTCTTCGGCTgtaatgttaaa-3′ (SEQ ID NO:6) edited the BCRP gene.
- FIG. 3 presents edited BCRP loci in two founder animals. One animal had a 588 bp deletion in exon 7, and the second animal had a 696 bp deletion in exon 7. These deletions disrupt the reading frame of the BCRP coding region.
- ZFNs that target and cleave the Pten locus in rats were designed and tested for activity essentially as described above in Example 1.
- An active pair of ZFNs was identified.
- the DNA binding sites were 5′-CCCCAGTTTGTGGTCtgcca-3′ (SEQ ID NO:7) and 5′-gcTAAAGGTGAAGATCTA-3′ (SEQ ID NO:8).
- polyadenylated mRNA encoding the active pair may be microinjected into rat embryos and the resultant embryos may be analyzed as described in Examples 1 and 2. Accordingly, the Pten locus may be edited to contain a deletion or an insertion such that the coding region is disrupted and no functional protein is made.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention provides genetically modified animals and cells comprising edited chromosomal sequences involved in tumor suppression. In particular, the animals or cells are generated using a zinc finger nuclease-mediated editing process. The invention also provides zinc finger nucleases that target chromosomal sequence involved in tumor suppression and the nucleic acids encoding the zinc finger nucleases. Also provided are methods of assessing the effects of agents in genetically modified animals and cells comprising edited chromosomal sequences involved in tumor suppression.
Description
- This application claims the priority of U.S. provisional application No. 61/343,287, filed Apr. 26, 2010, U.S. provisional application No. 61/323,702, filed Apr. 13, 2010, U.S. provisional application No. 61/323,719, filed Apr. 13, 2010, U.S. provisional application No. 61/323,698, filed Apr. 13, 2010, U.S. provisional application No. 61/309,729, filed Mar. 2, 2010, U.S. provisional application No. 61/308,089, filed Feb. 25, 2010, U.S. provisional application No. 61/336,000, filed Jan. 14, 2010, U.S. provisional application No. 61/263,904, filed Nov. 24, 2009, U.S. provisional application No. 61/263,696, filed Nov. 23, 2009, U.S. provisional application No. 61/245,877, filed Sep. 25, 2009, U.S. provisional application No. 61/232,620, filed Aug. 10, 2009, U.S. provisional application No. 61/228,419, filed Jul. 24, 2009, and is a continuation in part of U.S. non-provisional application Ser. No. 12/592,852, filed Dec. 3, 2009, which claims priority to U.S. provisional 61/200,985, filed Dec. 4, 2008 and U.S. provisional application 61/205,970, filed Jan. 26, 2009, all of which are hereby incorporated by reference in their entirety.
- The invention generally relates to genetically modified animals or cells comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression. In particular, the invention relates to the use of a zinc finger nuclease-mediated process to edit chromosomal sequences encoding proteins involved in tumor suppression.
- Tumor suppressors help regulate cell division and cell death. Mutations in tumor suppressor genes can lead to abnormal cell division and uncontrolled cell division, which is responsible for many types of cancer. This influx of cells in the body can grow into tissues, leading to specific cancers associated with various organs. Additionally, uncontrolled cell growth is responsible for blood cancer, such as leukemia. Half of all men and one-third of all women in the United States will develop cancer during their lifetimes. While research is being done to develop with new treatments for cancer, new treatment strategies and therapies are needed.
- The vast majority of drugs (approximately 91%) fail to successfully proceed through the three phases of drug testing in humans. Several drugs fail do so because of unforeseen toxicology in human patients, despite the fact that all of these drugs had been tested in animal models and were found to be safe. This is because toxicology testing is performed in animals, and animal proteins differ from the orthologous proteins in humans.
- What is needed are animals that are mutated for the genes involved in tumor suppression processes, including knockouts, multiple mutant lines (double knockouts, triple knockouts, etc.) and/or over-expression of alleles that either cause disease or are associated with disease in humans, as well as “humanized” animals that express or over-express human homologues of relevant genes in animals. Such animals could serve as research tools to develop and/or test new treatments for cancer.
- One aspect of the present disclosure encompasses a genetically modified animal comprising at least one edited chromosomal sequence encoding a sequence involved in tumor suppression.
- A further aspect provides a non-human embryo comprising at least one RNA molecule encoding a zinc finger nuclease that recognizes a chromosomal sequence encoding a protein involved in tumor suppression, and, optionally, at least one donor polynucleotide comprising a sequence encoding a protein involved in tumor suppression.
- Another aspect provides a genetically modified cell comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression.
- An alternate aspect provides a zinc finger nuclease comprising (a) a zinc finger DNA binding domain that binds a sequence having at least about 80% sequence identity to a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8; and (b) a cleavage domain.
- A further aspect provides a nucleic acid sequence recognized by a zinc finger nuclease. The nucleic acid sequence has at least about 80% sequence identity to a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8.
- Yet another aspect encompasses a method for assessing the therapeutic effect and/or toxicity of an agent. The method comprises (a) contacting a first genetically modified animal comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppressor with the agent; (b) measuring a therapeutic and/or toxicity response in the first animal; and (c) comparing the response in (b) to results obtained from a second genetically modified animal comprising the same edited chromosomal sequence encoding a protein involved in tumor suppression, wherein the second animal is not contacted with the agent.
- Other aspects and features of the disclosure are described more thoroughly below.
- The application file contains at least one figure executed in color. Copies of this patent application publication with color figures will be provided by the Office upon request and payment of the necessary fee.
-
FIG. 1 illustrates editing of the p53 locus in rats. Presented is a Cel-1 assay in which the presence of cleavage products indicated editing of the p53 gene. -
FIG. 2 illustrates knockout of the p53 gene in rats. Presented are Western blots of cytoplasmic and nuclear lysates of kidney (K) and liver (L) samples from wild-type (WT 731RP) and p53 knockout (KO 733RP) animals. The relative locations p53 protein and actin protein are indicated to the right of each image. -
FIG. 3 presents the DNA sequences of edited BCRP loci in two animals. (A) Shows a region of the rat BCRP locus (SEQ ID NO:1) comprising a 588 bp deletion in exon 7. (B) Presents a region of the rat BCRP locus (SEQ ID NO:2) comprising a 696 bp deletion in exon 7. The exon sequence is shown in green; the target site is presented in yellow, and the deletions are shown in dark blue. - The present disclosure provides a genetically modified animal or animal cell comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression. The edited chromosomal sequence may be (1) inactivated, (2) modified, or (3) comprise an integrated sequence. An inactivated chromosomal sequence is altered such that a functional protein is not made. Thus, a genetically modified animal comprising an inactivated chromosomal sequence may be termed a “knock-out” or a “conditional knock-out.” Similarly, a genetically modified animal comprising an integrated sequence may be termed a “knock-in” or a “conditional knock-in.” As detailed below, a knock-in animal may be a humanized animal. Furthermore, a genetically modified animal comprising a modified chromosomal sequence may comprise a targeted point mutation(s) or other modification such that an altered protein product is produced. The chromosomal sequence encoding a protein involved in tumor suppression generally is edited using a zinc finger nuclease-mediated process. Briefly, the process comprises introducing into an embryo or cell at least one RNA molecule encoding a targeted zinc finger nuclease and, optionally, at least one accessory polynucleotide. The method further comprises incubating the embryo or cell to allow expression of the zinc finger nuclease, wherein a double-stranded break introduced into the targeted chromosomal sequence by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process or a homology-directed DNA repair process. The method of editing chromosomal sequences involved in tumor suppression using targeted zinc finger nuclease technology is rapid, precise, and highly efficient.
- One aspect of the present disclosure provides a genetically modified animal in which at least one chromosomal sequence associated with tumor suppression has been edited. For example, the edited chromosomal sequence may be inactivated such that the sequence is not transcribed and/or a functional protein is not produced. Alternatively, the chromosomal sequence may be edited such that the regulation of expression of the protein is altered. For instance, the chromosomal sequence may be modified such that the protein associated with tumor suppression is over-produced. The edited chromosomal sequence may also be modified such that it codes for an altered tumor suppressor protein. For example, the chromosomal sequence may be modified such that at least one nucleotide is changed and the expressed protein comprises at least one changed amino acid residue (i.e., comprises a missense mutation). Furthermore, the edited chromosomal sequence may comprise a chromosomally integrated sequence encoding a protein associated with tumor suppression. The chromosomally integrated sequence may encode an endogenous protein associated with tumor suppression normally found in the animal, or the integrated sequence may encode an exogenous orthologous protein associated with tumor suppression, or combinations of both. The genetically modified animal disclosed herein may be heterozygous for the edited chromosomal sequence. Alternatively, the genetically modified animal may be homozygous for the edited chromosomal sequence.
- In one embodiment, the genetically modified animal may comprise at least one inactivated chromosomal sequence encoding a protein associated with tumor suppression. The inactivated chromosomal sequence may include a deletion mutation (i.e., deletion of one or more nucleotides), an insertion mutation (i.e., insertion of one or more nucleotides), or a point mutation (i.e., substitution of a single nucleotide for another nucleotide). The deletion, insertion, or point mutation may lead to frame shift and/or splice site mutations such that at least one premature stop codon is introduced. As a consequence of the mutation, the targeted chromosomal sequence is inactivated and a functional protein is not produced. The inactivated chromosomal sequence comprises no exogenously introduced sequence. Such an animal may be termed a “knock-out.” Also included herein are genetically modified animals in which two, three, or more chromosomal sequences encoding proteins associated with tumor suppression are inactivated.
- In another embodiment, the edited chromosomal sequence may be modified such that it codes for an altered protein associated with tumor suppression. The chromosomal sequence may be modified such that at least one nucleotide is changed and the expressed protein comprises at least one changed amino acid residue (i.e., a missense mutation). The chromosomal sequence may be modified to comprise more than one missense mutation such that more than one amino acid is changed. Additionally, the chromosomal sequence may be modified to have a three nucleotide deletion or insertion such that the expressed protein comprises a single amino acid deletion or insertion, provided such a protein is functional. The modified protein associated with tumor suppression may have altered substrate specificity, altered enzyme activity, altered kinetic rates, and so forth. In some embodiments, the modified protein comprises at least one modification such that the altered version of the protein provides tumor suppression activity. In other embodiments, the modified protein comprises at least one modification such that the altered version of the protein attenuates tumor suppression.
- In a further embodiment, the genetically modified animal may comprise at least one chromosomally integrated sequence encoding a protein associated with tumor suppression. Integration of the sequence encoding a protein associated with tumor suppression may be random or it may be targeted. For example, an exogenous sequence encoding an orthologous or an endogenous protein associated with tumor suppression may be integrated into a chromosomal sequence encoding a protein associated with tumor suppression (or another protein) such that the endogenous chromosomal sequence is inactivated, but wherein the exogenous sequence encoding the orthologous or endogenous protein may be expressed. In such a case, the sequence encoding the orthologous or endogenous protein may be operably linked to an endogenous promoter control sequence. Alternatively, the promoter control sequence may be native to the exogenous sequence or the exogenous sequence may be operably linked to a heterologous promoter control sequence. The exogenous sequence encoding the orthologous or endogenous protein may be such that the protein associated with tumor suppression is over-produced, or the tissue-specific or temporal expression of the protein is altered, or a combination thereof. Alternatively, an exogenous sequence encoding an orthologous or endogenous protein may be integrated into a chromosomal sequence without affecting expression of an endogenous chromosomal sequence. For example, an exogenous sequence encoding a protein associated with tumor suppression may be integrated into a “safe harbor” locus, such as the Rosa26 locus, HPRT locus, or AAVS1 locus, wherein the exogenous sequence encoding the orthologous or endogenous protein may be expressed or over-expressed. An animal comprising a chromosomally integrated sequence encoding a protein associated with tumor suppression may be called a “knock-in,” and it should be understood that in such an iteration of the animal, no selectable marker is present. The sequence encoding a protein associated with tumor suppression also may be modified to include a tag or reporter. Suitable reporters include selectable markers such as cloramphenicol acetyltransferase (CAT) and neomycin phosphotransferase (neo), and fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein, or any genetically engineered variant thereof that improves the reporter performance. Non-limiting examples of known such FP variants include EGFP, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet).
- The chromosomally integrated sequence encoding a protein associated with tumor suppression may encode the wild-type form of the protein. Alternatively, the chromosomally integrated sequence encoding a protein associated with tumor suppression may comprise at least one modification such that an altered version of the protein is produced. In some embodiments, the chromosomally integrated sequence encoding a protein associated with tumor suppression comprises at least one modification such that the altered version of the protein enhances tumor suppression. In other embodiments, the chromosomally integrated sequence encoding a protein associated with tumor suppression comprises at least one modification such that the altered version of the protein attenuates tumor suppression.
- In an additional embodiment, the genetically modified animal may be a “humanized” animal comprising at least one chromosomally integrated sequence encoding a functional tumor suppressor protein. The functional human protein may have no corresponding ortholog in the genetically modified animal. Alternatively, the wild-type animal from which the genetically modified animal is derived may comprise an ortholog corresponding to the human tumor suppressor protein. In this case, the orthologous sequence in the “humanized” animal is inactivated such that no functional protein is made and the “humanized” animal comprises at least one chromosomally integrated sequence encoding the human tumor suppressor protein. For example, a humanized animal may comprise an inactivated p53 sequence and a chromosomally integrated sequence encoding human p53 protein. Those of skill in the art appreciate that “humanized” animals may be generated by crossing a knock-out animal with a knock-in animal comprising the chromosomally integrated sequence.
- In yet another embodiment, the genetically modified animal may comprise at least one edited chromosomal sequence encoding a protein associated with tumor suppression such that the expression pattern of the protein associated with tumor suppression is altered. For example, regulatory regions controlling the expression of the protein, such as a promoter or transcription binding site, may be altered such that the protein associated with tumor suppression is over-produced, or the tissue-specific or temporal expression of the protein is altered, or a combination thereof. Alternatively, the expression pattern of the protein associated with tumor suppression may be altered using a conditional knockout system. A non-limiting example of a conditional knockout system includes a Cre-lox recombination system. A Cre-lox recombination system comprises a Cre recombinase enzyme, a site-specific DNA recombinase that can catalyze the recombination of a nucleic acid sequence between specific sites (lox sites) in a nucleic acid molecule. Methods of using this system to produce temporal and tissue specific expression are known in the art. In general, a genetically modified animal is generated with lox sites flanking a chromosomal sequence, such as a chromosomal sequence encoding a protein associated with tumor suppression. The genetically modified animal comprising the lox-flanked chromosomal sequence encoding a protein associated with tumor suppression may then be crossed with another genetically modified animal expressing Cre recombinase. Progeny animals comprising the lox-flanked chromosomal sequence and the Cre recombinase are then produced, and the lox-flanked chromosomal sequence encoding a protein associated with tumor suppression is recombined, leading to deletion or inversion of the chromosomal sequence encoding a protein associated with tumor suppression. Expression of Cre recombinase may be temporally and conditionally regulated to effect temporally and conditionally regulated recombination of the chromosomal sequence encoding a protein associated with tumor suppression.
- Tumor suppression genes are genes whose protein products protect a cell from one step on the path to cancer. A mutation in a tumor suppressor gene may cause a loss or reduction in the protective function of its protein product, thereby increasing the probability that a tumor will form, leading to cancer, usually in combination with other genetic changes. The proteins encoded by tumor suppressor genes have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes both. Tumor suppressor proteins are involved in the repression of genes essential for the continuing cell cycle; coupling the cell cycle to DNA damage so that the cell cycle can continue; initiating apoptosis in the cell if the damage cannot be repaired; and cell adhesion to prevent tumors from dispersing, blocking a loss of contact inhibition, and inhibiting metastasis.
- Mutations in tumor suppressor genes can lead to various types of cancer, including but not limited to Retinoblastoma, Human Papilloma Virus, Wilms Tumor,
Neurofibromatosis Type 1, Neurofibromatosis Type 2, familial adenomatous polyposis, Colon Cancer, Von Hippel-Lindau syndrome, Li-Fraumeni Syndrome, Familial Juvenile Polyposis syndrome, Familial Breast Cancer, Cowden Syndrome, Peutz-Jeghers Syndrome, Hereditary NonpolyposisColon Cancer Type 1, Hereditary Nonpolyposis Colon Cancer Type 2, Familial diffuse-type Gastic Cancer, Familial Melanoma, Gorlin Syndrome, MultipleEndocrine Neoplasia Type 1, and other tumor-related diseases. - The present disclosure comprises editing of any chromosomal sequences that encodes a protein associated with tumor suppression. The proteins associated with tumor suppression are typically selected based on an experimental association of the protein of interest with a cancer. For example, the production rate or circulating concentration of a protein associated with tumor suppression may be elevated or depressed in a population having cancer relative to a population not having cancer. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the proteins associated with tumor suppression may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).
- By way of example, proteins involved in tumor suppression and their encoding chromosomal sequences may comprise, but is not limited to, TNF (tumor necrosis factor (TNF superfamily, member 2)), TP53 (tumor protein p53), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)), FN1 (fibronectin 1), TSC1 (tuberous sclerosis 1), PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), PTEN (phosphatase and tensin homolog), PCNA (proliferating cell nuclear antigen), COL18A1 (collagen, type XVIII, alpha 1), TSSC4 (tumor suppressing subtransferable candidate 4), JUN (jun oncogene), MAPK8 (mitogen-activated protein kinase 8), TGFB1 (transforming growth factor, beta 1), IL6 (interleukin 6 (interferon, beta 2)), IFNG (interferon, gamma), BRCA1 (breast cancer 1, early onset), TSPAN32 (tetraspanin 32), BCL2 (B-cell CLL/lymphoma 2), NF2 (neurofibromin 2 (merlin)), GJB1 (gap junction protein, beta 1, 32 kDa), MAPK1 (mitogen-activated protein kinase 1), CD44 (CD44 molecule (Indian blood group)), PGR (progesterone receptor), TNS1 (tensin 1), PROK1 (prokineticin 1), SIAH1 (seven in absentia homolog 1 (Drosophila)), ENG (endoglin), TP73 (tumor protein p73), APC (adenomatous polyposis coli), BAX (BCL2-associated X protein), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)), VHL (von Hippel-Lindau tumor suppressor), FHIT (fragile histidine triad gene), NFKB1 (nuclear factor of kappa light polypeptide gene enhancer in B-cells 1), IFNA1 (interferon, alpha 1), TGFBR1 (transforming growth factor, beta receptor 1), PRKCD (protein kinase C, delta), TGIF1 (TGFB-induced factor homeobox 1), DLC1 (deleted in liver cancer 1), SLC22A18 (solute carrier family 22, member 18), VEGFA (vascular endothelial growth factor A), MME (membrane metallo-endopeptidase), IL3 (interleukin 3 (colony-stimulating factor, multiple)), MKI67 (antigen identified by monoclonal antibody Ki-67), HSPD1 (heat shock 60 kDa protein 1 (chaperonin)), HSPB1 (heat shock 27 kDa protein 1), HSP90B2P (heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonic defect)), ZFYVE9 (zinc finger, FYVE domain containing 9), TERT (telomerase reverse transcriptase), PML (promyelocytic leukemia), SKP2 (S-phase kinase-associated protein 2 (p45)), CYCS (cytochrome c, somatic), MAPK10 (mitogen-activated protein kinase 10), PAX7 (paired box 7), YAP1 (Yes-associated protein 1), PARP1 (poly (ADP-ribose) polymerase 1), MIR34A (microRNA 34a), PRKCA (protein kinase C, alpha), FAS (Fas (TNF receptor superfamily, member 6)), SYK (spleen tyrosine kinase), GSK3B (glycogen synthase kinase 3 beta), PRKCE (protein kinase C, epsilon), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), ABCB1 (ATP-binding cassette, sub-family B (MDR/TAP), member 1), NFKBIA (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), RUNX1 (runt-related transcription factor 1), PRKCG (protein kinase C, gamma), RELA (v-rel reticuloendotheliosis viral oncogene homolog A (avian)), PLAU (plasminogen activator, urokinase), BTK (Bruton agammaglobulinemia tyrosine kinase), PRKCB (protein kinase C, beta), CSF1 (colony stimulating factor 1 (macrophage)), POMC (proopiomelanocortin), CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), ROCK1 (Rho-associated, coiled-coil containing protein kinase 1), KDR (kinase insert domain receptor (a type III receptor tyrosine kinase)), NPM1 (nucleophosmin (nucleolar phosphoprotein B23, numatrin)), ROCK2 (Rho-associated, coiled-coil containing protein kinase 2), PRKAB1 (protein kinase, AMP-activated, beta 1 non-catalytic subunit), BAK1 (BCL2-antagonist/killer 1), AURKA (aurora kinase A), NTN1 (netrin 1), FLT1 (fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)), NBN (nibrin), DNM3 (dynamin 3), PRDM10 (PR domain containing 10), PAX5 (paired box 5), EIF4G1 (eukaryotic translation initiation factor 4 gamma, 1), KAT2B (K(lysine)acetyltransferase 2B), TIMP3 (TIMP metallopeptidase inhibitor 3), CCL22 (chemokine (C—C motif) ligand 22), GRIN2B (glutamate receptor, ionotropic, N-methyl D-aspartate 2B), CD81 (CD81 molecule), CCL27 (chemokine (C—C motif) ligand 27), MAPK11 (mitogen-activated protein kinase 11), DKK1 (dickkopf homolog 1 (Xenopus laevis)), HYAL1 (hyaluronoglucosaminidase 1), CTSL1 (cathepsin L1), PKD1 (polycystic kidney disease 1 (autosomal dominant)), BUB1B (budding uninhibited by benzimidazoles 1 homolog beta (yeast)), MPP1 (membrane protein, palmitoylated 1, 55 kDa), SIAH2 (seven in absentia homolog 2 (Drosophila)), DUSP13 (dual specificity phosphatase 13), CCL21 (chemokine (C—C motif) ligand 21), RTN4 (reticulon 4), SMO (smoothened homolog (Drosophila)), CCL19 (chemokine (C—C motif) ligand 19), CSTF2 (cleavage stimulation factor, 3\′ pre-RNA, subunit 2, 64 kDa), RSF1 (remodeling and spacing factor 1), EZH2 (enhancer of zeste homolog 2 (Drosophila)), AK1 (adenylate kinase 1), CKM (creatine kinase, muscle), HYAL3 (hyaluronoglucosaminidase 3), ALOX15B (arachidonate 15-lipoxygenase, type B), PAG1 (phosphoprotein associated with glycosphingolipid microdomains 1), MIR21 (microRNA 21), S100A2 (S100 calcium binding protein A2), HYAL2 (hyaluronoglucosaminidase 2), CSTF1 (cleavage stimulation factor, 3\′ pre-RNA, subunit 1, 50 kDa), PCGF2 (polycomb group ring finger 2), THSD1 (thrombospondin, type I, domain containing 1), HOPX (HOP homeobox), SLC5A8 (solute carrier family 5 (iodide transporter), member 8), EMB (embigin homolog (mouse)), PAX9 (paired box 9), ARMCX3 (armadillo repeat containing, X-linked 3), ARMCX2 (armadillo repeat containing, X-linked 2), ARMCX1 (armadillo repeat containing, X-linked 1), RASSF4 (Ras association (RaIGDS/AF-6) domain family member 4), MIR34B (microRNA 34b), MIR205 (microRNA 205), RB1 (retinoblastoma 1), DYT10 (dystonia 10), CDKN2A (cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)), CDKN1A (cyclin-dependent kinase inhibitor 1A (p21, Cip1)), CCND1 (cyclin D1), AKT1 (v-akt murine thymoma viral oncogene homolog 1), MYC (v-myc myelocytomatosis viral oncogene homolog (avian)), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa), MDM2 (Mdm2 p53 binding protein homolog (mouse)), SERPINB5 (serpin peptidase inhibitor, clade B (ovalbumin), member 5), EGF (epidermal growth factor (beta-urogastrone)), FOS (FBJ murine osteosarcoma viral oncogene homolog), NOS2 (nitric oxide synthase 2, inducible), CDK4 (cyclin-dependent kinase 4), SOD2 (superoxide dismutase 2, mitochondrial), SMAD3 (SMAD family member 3), CDKN1B (cyclin-dependent kinase inhibitor 1B (p27, Kip1)), SOD1 (superoxide dismutase 1, soluble), CCNA2 (cyclin A2), LOX (lysyl oxidase), SMAD4 (SMAD family member 4), HGF (hepatocyte growth factor (hepapoietin A; scatter factor)), THBS1 (thrombospondin 1), CDK6 (cyclin-dependent kinase 6), ATM (ataxia telangiectasia mutated), STAT3 (signal transducer and activator of transcription 3 (acute-phase response factor)), HIF1A (hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)), IGF1R (insulin-like growth factor 1 receptor), MTOR (mechanistic target of rapamycin (serine/threonine kinase)), TSC2 (tuberous sclerosis 2), CDC42 (cell division cycle 42 (GTP binding protein, 25 kDa)), ODC1 (ornithine decarboxylase 1), SPARC (secreted protein, acidic, cysteine-rich (osteonectin)), HDAC1 (histone deacetylase 1), CDK2 (cyclin-dependent kinase 2), BARD1 (BRCA1 associated RING domain 1), CDH1 (cadherin 1, type 1, E-cadherin (epithelial)), EGR1 (early growth response 1), INSR (insulin receptor), IRF1 (interferon regulatory factor 1), PHB (prohibitin), PXN (paxillin), HSPA4 (heat shock 70 kDa protein 4), TYR (tyrosinase (oculocutaneous albinism IA)), CAV1 (caveolin 1, caveolae protein, 22 kDa), CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)), FOXO3 (forkhead box O3), HDAC9 (histone deacetylase 9), FBXW7 (F-box and WD repeat domain containing 7), FOXO1 (forkhead box O1), E2F1 (E2F transcription factor 1), STK11 (serine/threonine kinase 11), BMP2 (bone morphogenetic protein 2), HSP90AA1 (heat shock protein 90 kDa alpha (cytosolic), class A member 1), HNF4A (hepatocyte nuclear factor 4, alpha), CAMK2G (calcium/calmodulin-dependent protein kinase II gamma), TP53BP1 (tumor protein p53 binding protein 1), CRYAB (crystallin, alpha B), HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme A reductase), PLAUR (plasminogen activator, urokinase receptor), MCL1 (myeloid cell leukemia sequence 1 (BCL2-related)), NOTCH1 (Notch homolog 1, translocation-associated (Drosophila)), RASSF1 (Ras association (RaIGDS/AF-6) domain family member 1), GSN (gelsolin), CADM1 (cell adhesion molecule 1), ATF2 (activating transcription factor 2), IFNB1 (interferon, beta 1, fibroblast), DAPK1 (death-associated protein kinase 1), CHFR (checkpoint with forkhead and ring finger domains), KITLG (KIT ligand), NDUFA13 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13), DPP4 (dipeptidyl-peptidase 4), GLB1 (galactosidase, beta 1), IKZF1 (IKAROS family zinc finger 1 (Ikaros)), ST5 (suppression of tumorigenicity 5), TGFA (transforming growth factor, alpha), EIF4EBP1 (eukaryotic translation initiation factor 4E binding protein 1), TGFBR2 (transforming growth factor, beta receptor II (70/80 kDa)), EIF2AK2 (eukaryotic translation initiation factor 2-alpha kinase 2), GJA1 (gap junction protein, alpha 1, 43 kDa), MYD88 (myeloid differentiation primary response gene (88)), IF127 (interferon, alpha-inducible protein 27), RBMX (RNA binding motif protein, X-linked), EPHA1 (EPH receptor A1), TWSG1 (twisted gastrulation homolog 1 (Drosophila)), H2AFX (H2A histone family, member X), LGALS3 (lectin, galactoside-binding, soluble, 3), MUC3A (mucin 3A, cell surface associated), ILK (integrin-linked kinase), APAF1 (apoptotic peptidase activating factor 1), MAOA (monoamine oxidase A), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)), EIF2S1 (eukaryotic translation initiation factor 2, subunit 1 alpha, 35 kDa), PER2 (period homolog 2 (Drosophila)), IGFBP7 (insulin-like growth factor binding protein 7), KDM5B (lysine (K)-specific demethylase 5B), SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), NME1 (non-metastatic cells 1, protein (NM23A) expressed in), F2RL1 (coagulation factor II (thrombin) receptor-like 1), ZFP36 (zinc finger protein 36, C3H type, homolog (mouse)), HSPA8 (heat shock 70 kDa protein 8), WNTSA (wingless-type MMTV integration site family, member 5A), ITGB4 (integrin, beta 4), RARB (retinoic acid receptor, beta), VEGFC (vascular endothelial growth factor C), CCL20 (chemokine (C—C motif) ligand 20), EPHB2 (EPH receptor B2), CSNK2A1 (casein kinase 2, alpha 1 polypeptide), PSMD9 (proteasome (prosome, macropain) 26S subunit, non-ATPase, 9), SERPINB2 (serpin peptidase inhibitor, clade B (ovalbumin), member 2), RHOB (ras homolog gene family, member B), DUSP6 (dual specificity phosphatase 6), CDKN1C (cyclin-dependent kinase inhibitor 1C (p57, Kip2)), SLIT2 (slit homolog 2 (Drosophila)), CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein)), UBC (ubiquitin C), STS (steroid sulfatase (microsomal), isozyme S), FST (follistatin), KRT1 (keratin 1), EIF6 (eukaryotic translation initiation factor 6), JUP (junction plakoglobin), HDAC4 (histone deacetylase 4), NEDD4 (neural precursor cell expressed, developmentally down-regulated 4), KRT14 (keratin 14), GLI2 (GLI family zinc finger 2), MYH11 (myosin, heavy chain 11, smooth muscle), MAPKAPK5 (mitogen-activated protein kinase-activated protein kinase 5), MAD1L1 (MAD1 mitotic arrest deficient-like 1 (yeast)), TNFAIP3 (tumor necrosis factor, alpha-induced protein 3), WEE1 (WEE1 homolog (S. pombe)), BTRC (beta-transducin repeat containing), NKX3-1 (NK3 homeobox 1), GPC3 (glypican 3), CREB3 (cAMP responsive element binding protein 3), PLCB3 (phospholipase C, beta 3 (phosphatidylinositol-specific)), DMPK (dystrophia myotonica-protein kinase), BLNK (B-cell linker), PPIA (peptidylprolyl isomerase A (cyclophilin A)), DAB2 (disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila)), KLF4 (Kruppel-like factor 4 (gut)), RUNX3 (runt-related transcription factor 3), FLG (filaggrin), IVL (involucrin), COTS (chaperonin containing TCP1, subunit 5 (epsilon)), LRPAP1 (low density lipoprotein receptor-related protein associated protein 1), IGF2R (insulin-like growth factor 2 receptor), PERI (period homolog 1 (Drosophila)), BIK (BCL2-interacting killer (apoptosis-inducing)), PSMC4 (proteasome (prosome, macropain) 26S subunit, ATPase, 4), USF2 (upstream transcription factor 2, c-fos interacting), GAS1 (growth arrest-specific 1), LAMP2 (lysosomal-associated membrane protein 2), PSMD10 (proteasome (prosome, macropain) 26S subunit, non-ATPase, 10), IL24 (interleukin 24), GADD45G (growth arrest and DNA-damage-inducible, gamma), ARHGAP1 (Rho GTPase activating protein 1), CLDN1 (claudin 1), ANXA7 (annexin A7), CHN1 (chimerin (chimaerin) 1), TXNIP (thioredoxin interacting protein), PEG3 (paternally expressed 3), EIF3A (eukaryotic translation initiation factor 3, subunit A), CASC5 (cancer susceptibility candidate 5), TCF4 (transcription factor 4), CSNK2A2 (casein kinase 2, alpha prime polypeptide), CSNK2B (casein kinase 2, beta polypeptide), CRY1 (cryptochrome 1 (photolyase-like)), CRY2 (cryptochrome 2 (photolyase-like)), EIF4G2 (eukaryotic translation initiation factor 4 gamma, 2), LOXL2 (lysyl oxidase-like 2), PSMD13 (proteasome (prosome, macropain) 26S subunit, non-ATPase, 13), ANP32A (acidic (leucine-rich) nuclear phosphoprotein 32 family, member A), COL4A3 (collagen, type IV, alpha 3 (Goodpasture antigen)), SCGB1A1 (secretoglobin, family 1A, member 1 (uteroglobin)), BNIP3L (BCL2/adenovirus E1B 19 kDa interacting protein 3-like), MCC (mutated in colorectal cancers), EFNB3 (ephrin-B3), RBBP8 (retinoblastoma binding protein 8), PALB2 (partner and localizer of BRCA2), HBP1 (HMG-box transcription factor 1), MRPL28 (mitochondrial ribosomal protein L28), KDM5A (lysine (K)-specific demethylase 5A), QSOX1 (quiescin Q6 sulfhydryl oxidase 1), ZFR (zinc finger RNA binding protein), MN1 (meningioma (disrupted in balanced translocation) 1), SMYD4 (SET and MYND domain containing 4), USP7 (ubiquitin specific peptidase 7 (herpes virus-associated)), STK4 (serine/threonine kinase 4), THY1 (Thy-1 cell surface antigen), PTPRG (protein tyrosine phosphatase, receptor type, G), E2F6 (E2F transcription factor 6), STX11 (syntaxin 11), CDC42BPA (CDC42 binding protein kinase alpha (DMPK-like)), MYOCD (myocardin), DAP (death-associated protein), LOXL1 (lysyl oxidase-like 1), RNF139 (ring finger protein 139), HTATIP2 (HIV-1 Tat interactive protein 2, 30 kDa), AIM1 (absent in melanoma 1), BCCIP (BRCA2 and CDKN1A interacting protein), LOXL4 (lysyl oxidase-like 4), WWC1 (WW and C2 domain containing 1), LOXL3 (lysyl oxidase-like 3), CENPN (centromere protein N), TNS4 (tensin 4), SIK1 (salt-inducible kinase 1), PCGF6 (polycomb group ring finger 6), PHLDA3 (pleckstrin homology-like domain, family A, member 3), IL32 (interleukin 32), LATS1 (LATS, large tumor suppressor, homolog 1 (Drosophila)), COMMD7 (COMM domain containing 7), CDHR2 (cadherin-related family member 2), LELP1 (late cornified envelope-like proline-rich 1), NCRNA00188 (non-protein coding RNA 188), and ENSG00000131023.
- Exemplary non-limiting examples of tumor suppression proteins include ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor 1a), HIF3a (hypoxia-inducible factor 1a), Met (met pronto-oncogene), HRG (histidine-rich glycoprotein), Bc12, PPAR(alpha) (peroxisome proliferator-activated receptor alpha), Ppar(gamma) (peroxisome proliferator-activated receptor gamma), WT1 (Wilmus Tumor 1), FGF1R (fibroblast growth factor 1 receptor) , FGF2R (fibroblast growth factor 1 receptor), FGF3R (fibroblast growth factor 3 receptor), FGF4R (fibroblast growth factor 4 receptor), FGF5R (fibroblast growth factor 5 receptor), CDKN2a (cyclin-dependent kinase inhibitor 2A), APC (adenomatous polyposis coli), Rb1 (retinoblastoma 1), MEN1 (multiple endocrine neoplasia1), VHL (von-Hippel-Lindau tumor suppressor), BRCA1 (breast cancer 1), BRCA2 (breast cancer 2), AR (androgen receptor), TSG101 (tumor susceptibility gene 101), Igf1(insulin-like growth factor 1), Igf2 (insulin-like growth factor 2), Igf 1R (insulin-like growth factor 1 receptor), Igf 2R (insulin-like growth factor 2 receptor), Bax (BCL-2 associated X protein), CASP 1 (Caspase 1), CASP 2 (Caspase 2), CASP 3 (Caspase 3), CASP 4(Caspase 4), CASP 6 (Caspase 6), CASP 7(Caspase 7), CASP 8 (Caspase 8), CASP 9 (Caspase 9), CASP 12 (Caspase 12), Kras (v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog), PTEN (phosphate and tensin homolog), BCRP (breast cancer receptor protein), p53, and combinations thereof.
- (i) ATM
- ATM, or ataxia telangiectasia mutated, provides instructions for making a protein, primarily located in the nucleus, which helps control the rate at which cells grow and divide. Additionally, ATM plays an important role in the normal development and activity of several body systems, including the nervous system and the immune system. ATM protein assists cells in recognizing damaged or broken DNA strands and coordinates DNA repair by activating enzymes that repair breaks in the DNA. The stability of the cell's genetic information is maintained by the efficient repair of damaged DNA. The ATM protein is of great interest in cancer research because of its central role in cell division and DNA repair.
- Mutations in the ATM gene have been shown to be involved in several conditions. Ataxia-telangiectasia is caused by one of several hundred mutations in the ATM gene. Those afflicted with this disorder have mutations in both copies of the ATM gene in a cell. Breast cancer has also been found to be associated with the ATM gene. Patients with at least one family member with ataxia-telangiectasia are thought to have an increased risk of developing breast cancer. The mutation in the ATM gene prevents many of the body's cells from correctly repairing damaged DNA. People who have only one copy of the ATM gene in each cell, due to a deletion, are also at an increased risk of developing breast cancer. Cells that are missing one copy of the ATM gene produce half of the normal amount of ATM protein which leads to inefficient repair of DNA damage, leading to the accumulation of mutations in other genes. This leads to the possibility of the development of cancerous tumors. It has also been shown that people who carry one mutated copy of the ATM gene in each cell may have an increased risk of developing several other types of cancers, including, stomach, bladder, pancreas, lung, and ovarian cancer.
- (ii) ATR
- ATR, or ataxia telangiectasia and Rad3 related, is a protein kinase. Mutations of the ATR gene are associated with Sekel syndrome, a rare autosomal recessive disorder characterized by growth retardation, microencephaly with mental retardation, and a characteristic “bird headed” facial appearance. ATR is thought to be involved in DNA replication and DNA repair.
- EGFR
- EGFR or epidermal growth factor receptor is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor. When EGFR binds to a ligand, receptor dimerization and tyrosine autophosphorylation is induced, leading to cell proliferation. Mutations in EGFR are associated with lung cancer.
- (iv) ERBB2
- ERBB2, or v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog, is commonly referred to as Her-2/neu. ERBB2 growth factor receptor is located on the cell surface, where it associates with similar receptors to form a complex. Growth factors bind to these similar receptors and trigger the receptor complex to relay signals inside the cell activating certain genes that promote cell growth. It is thought that ERBB2 plays a role in cell adhesion, cell specialization, and cell movement. Breast cancer is associated with the ERBB2 gene and amplification of the ERBB2 gene is found in about 25% of breast cancers. The mutations in the ERBB2 gene are somatic mutations, thus, they are not inherited. One mechanism of somatic mutations is where DNA replicates in preparation for cell division resulting in multiple copies of the gene on a chromosome. Tumors can form as a result of multiple gene copies. Amplification of ERBB2 has also been linked to other types of cancer including ovarian, brain, stomach, and lung cancers.
- (v) ERBB3
- ERBB3 is also known as v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 is a member of the epidermal growth factor receptor family of receptor tyrosine kinases. Amplification of this gene and overexpression of the ERBB3 protein have been linked to numerous cancers, including prostate, bladder, and breast tumors. ERBB3 binds to and is activated by neuregulins and NTAK. Mutations and defects in the ERBB3 gene are the cause of the lethal congenital contracture syndrome type 2, also referred to as Israeli Bedouin multiple contracture syndrome type A. Israeli Bedouin multiple contracture syndrome type A is characterized by multiple joint contractures, anterior horn atrophy in the spinal chord, and a distended bladder.
- (vi) ERBB4
- ERBB4 is also known as v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 is related to ERBB2 and ERBB3 and serves a similar function.
- (vii)
Notch 1 -
Notch 1 is a member of theType 1 transmembrane protein family and shares structural characteristics including an extracellular domain consisting of multiple epidermal growth factor-like repeats (EGF) and an intracellular domain consisting of multiple, different domain types. Notch proteins play a role in a variety of developmental processes by affecting the cell fate decisions. Notch genes have a signaling network that is an intracellular pathway which regulates interaction between physically adjacent cells. Defects inNotch 1 are a cause of bicuspid aortic valve, a common defect in the aortic valve where three heart leaflets are present instead of two. In rare cases, mutations in this gene can lead to restricted blood flow resulting in hypoplastic left heart syndrome. Aortic valve disease and T-cell acute lymphoblastic leukemia are associated with mutations in theNotch 1 gene. - (viii) Notch 2
- Notch 2 is related to
Notch 1, as both proteins are members of the Notch family of receptor proteins. Notch 2 has five ligands: Jagged 1, Jagged 2, Delta-like 1, Delta-like 3, and Delta-like 4. The Notch 2 protein and its ligands send signals that are important prior to birth, with research indicating that signals triggered by the interaction between Notch 2 and its ligands contribute to the development of cells destined to be a part of the heart, liver, kidney, teeth, and other structures in the growing embryo. Notch 2 is involved in tissue repair after birth. - Mutations in Notch 2 have been associated with Alagille syndrome. It is hypothesized that the mutations in Notch 2 probably result in a protein that is abnormally small or folded into an incorrect three-dimensional shape. Disrupted signaling in the Notc h2 gene is believed to cause development issues in the heart, liver, kidney and other parts of the body, resulting in the signs and symptoms of Alagille syndrome. Mutations in Notch 2 may be somatic mutations. These somatic mutations may lead to extra copies of the mutated gene, increasing gene activity. This increase in gene activity may lead to uncontrolled cell growth and cell division in the immune system cells, causing tumors.
- (ix) Notch 3
- Notch 3 receptor protein is located on the surface of muscle cells that surround blood vessels. Notch 3 receptor protein is specific to the arteries; however, the protein is not present in veins. Signals are sent by Notch 3 to the nucleus of the cell such that particular genes are activated within vascular smooth muscle cells. The Notch 3 receptors are though to be essential for the maintenance of healthy muscle cells in the brain's arteries.
- Notch 3 is thought to be responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) due to one of 150 known mutations in the gene. Almost all of the 150 mutations change a single amino acid in the Notch 3 receptor. Disruption of the function of Notch 3 may lead to apoptosis of cells and damage to vascular smooth muscle cells. This damage to the vascular smooth muscle tissue is thought to cause recurrent strokes and other symptoms of CADASIL.
- (x) Notch 4
- Notch 4 is related to
Notch 1, Notch 2, and Notch 3 and serves a similar function. - (xi) AKT1
- AKT1, or v-akt murine thymoma
viral oncogene homolog 1, is a serine-threonine protein kinase that is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and related AKT2 are activated by platelet-derived growth factor. Growth factor-induced neuronal survival is mediated by AKTs. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates the components of the apoptosis machinery. - Mutations in AKT1 are associated with colorectal cancer and an increased susceptibility to ovarian cancer and familial breast-
ovarian cancer type 1. Additionally, mutations in AKT1 have been linked to the susceptibility of schizophrenia. - (xii) AKT2
- AKT2, or v-akt murine thymona viral oncogene homolog 2, belongs to the subfamily of serine threonine kinases containing SH2-like domains. AKT2 was shown to be amplified and overexpressed in ovarian cancer cell lines and primary ovarian tumors. The overexpression of the AKT2 protein contributes to a malignant phenotype of human ductal pancreatic cancers. The AKT2 gene and protein is indicated in diabetes mellitus.
- (xiii) AKT3
- AKT3, or v-akt murine thymona viral oncogene homolog 3, encodes the AKT3 protein. The AKT3 gene is related to the AKT1 and AKT2 genes and mutations in the AKT3 gene caused overexpression of the AKT3 protein.
- (xiv) HIF1A and HIF1B
- HIF1A, hypoxia-
inducible factor 1, alpha subunit, and HIF1B, hypoxia-inducible factor 1, beta subunit, encode the HIF1A and HIF1B proteins, respectively. HIF is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau tumor suppressor protein leads to rapid degradation of the HIF1A protein. Polymorphisms in the HIF1A are associated with renal cell carcinoma phenotype. The use of immunohistochemical assessment of HIF1A can be used as a predictor of poor outcome may improve clinical decision making regarding adjuvant treatment of patients with lymph node negative breast carcinoma. - (xv) Met
- Met, or met pronto-oncogene (hepatocyte growth factor receptor), is a hepatocyte growth factor receptor that has tyrosine-kinase activity. The activation of MET after rearrangement with the TPR gene produces an oncogenic protein. MET has also been associated with gastric cancer, hepatocellular carcinoma, hereditary papillary renal carcinoma, and other related conditions. Genetic variations and mutations in MET may also be associated with susceptibility to autism type 9.
- (xvi) HRG
- HRG, or histidine-rich glycoprotein, contains two cysteine-like domains and is located in plasma and platelets. HRG can inhibit rosette formation and interacts with heparin, thrombospondin and plasminogen. There is a potential prothrombotic effect as exhibited by HRG's inhibition of fibrinolysis and the reduction of inhibition of coagulation. Mutations in HRG are thought to be the cause of thrombophilia due to histidine-rich glycoprotein deficiency.
- (xvii) Bc12
- Bc12 is a tumor suppressor protein related to p53. As a tumor suppressor, Bc12 is involved in the replication of cells. Mutations in Bc12 can lead to uncontrolled or abnormal cell replication.
- (xviii) Ppar(alpha)
- Ppar(alpha), or peroxisome proliferator-activated receptor alpha, is a nuclear receptor protein. Peroxisome proliferators include hypolipidemic drugs, herbicides, leukotriene antagonists, and plasticizers. Peroxisomes are subcellular organelles found in plants and animals that contain enzymes for respiration and for cholesterol and lipid metabolism. The action of peroxisomes is thought to be mediated via specific receptors, called PPARs, which affect the expression of target genes involved in cell proliferation, cell differentiation and in immune and inflammatory responses. Mutations in Ppar(alpha) have been linked to the susceptibility to hyperapobetalipoproteinemia.
- (xix) Ppar(gamma)
- Ppar(gamma), or peroxisome proliferator-activated receptor gamma, is another nuclear receptor. Ppar(gamma) regulates adipocyte differentiation that has been implicated in the pathology of numerous diseases such as obesity, diabetes, atherosclerosis, familial partial lipodystrophy type 3, and cancer. Mutations in Ppar(gamma) are associated with carotid intimal
medial thickness 1. Other genetic variations in PPARG can be associated with susceptibility toglioma type 1. Gliomas are central nervous system neoplasms derived from glial cells and comprise astrocytomas, glioblastoma, multiforme, oligodendrogliomas, and ependymomas. - (xx) WT1
- WT1, or
Wilmus tumor 1, is a transcription factor that regulates the activity of genes involved in cell growth and apoptosis. Wilmus tumor is a rare form of kidney cancer, where the individual has a mutation in one copy of the WT1 gene in every cell. In other types of Wilmus tumor, WT1 gene mutations are only present in tumor cells. The mutations in the WT1 genes are somatic, meaning they are not inherited. Mutations in the WT1 gene have also been linked to several other forms of cancer including lung, prostate, breast, and ovarian cancer, as well as leukemia, such as acute lymphoblastic leukemia, chronic myeloid leukemia, and childhood acute myeloid leukemia. Denys-Drash syndrome is also caused by a mutation in the WT1 gene. WT1 mutations are also thought to cause Frasier syndrome. The mutations disrupt the way the WT1 gene's instructions are used to make the protein resulting in a shortage of functional protein. - (xxi) FGF Receptor Family
- The FGF receptor family or fibroblast growth factor receptor family includes FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5. FGF receptors are involved in important processes such as cell division, regulation of cell growth and maturation, formation of blood vessels, wound healing, and embryonic development. Growth factors work with the FGF receptor proteins to signal chemical reactions within the cell that instruct the cell to undergo certain changes, such as maturing to take on special functions.
- The FGFR1 protein is thought to play a role in the development of the nervous system. Mutations in the FGFR1 gene are responsible for Kallmann syndrome, Pfeiffer syndrome, osteoglophonic dysplasia, and cancers such as pancreatic, esophageal, ovarian, testicular, breast, and head and neck cancers. Mutations in the FGFR2 gene have been lined to Apert syndrome, Beare-Stevenson cutis gyrate syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, lacrimo-auriculo-dento-digital (LADD) syndrome, and cancers such as prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, and head and neck cancers. FGFR3 gene mutations have been associated with Achondroplasia, Crouzonodermoskeletal syndrome, hypochondroplasia, Muenke syndrome, SADDAN, thanatophoric dysplasia, bladder cancer, and platyspondylic lethal skeletal dysplasia. Several types of cancers are associated with mutations in the FGFR4 gene, such as breast, colon, gastric, pancreatic, ovarian, head and neck, and prostate. These cancers are usually linked to a polymorphism in which glycine is replaced by arginine at position 338 in the protein's chain of amino acids.
- (xxii) CDKN2a
- CDKN2a, or cyclin-dependent kinase inhibitor 2A, encodes the CDKN2a protein. The CDKN2a gene generates several transcript variants which differ in their first exons, with at least three alternatively spliced variants encoding distinct proteins, two of which encode structurally related isoforms known to function as inhibitors of CDK4 kinase. This interaction with CDK4 and with CDK6 allows CDKN2a to act as a negative regulator of the proliferation of normal cells. Further, CDKN2a is capable of inducing cell cycle arrest in G1 and G2 phases and acts as a tumor suppressor. Genetic variations in CDKN2a may underlie susceptibility to uveal melanoma, which is the most common type of ocular malignant tumor. Mutations in CDKN2a are also linked to cutaneous malignant melanoma type 2, familiar atypical multiple mole melanoma-pancreatic carcinoma syndrome, melanoma-astrocytoma syndrome, Li Fraumeni syndrome, melanoma and neural system tumor syndrome, orolaryngeal cancer, pancreatic cancer, and melanoma syndrome.
- (xxiii) APC
- APC, is also referred to as adenomatous polyposis coli, plays a critical role in several cellular processes that determine whether a cell will develop into a tumor. The APC protein acts as a tumor suppressor, thus, it regulates the cell division cycle by keeping cells from growing and dividing too fast or in an uncontrolled way. Cell division, attachment and migration are all controlled, in part, by the APC protein. Mutations in the APC gene are associated with familial adenomatous polyposis, for which over 700 mutations have been identified. Cancers such as colorectal cancer, Turcot syndrome, colon cancer, and stomach cancer have been linked to mutations in the APC gene. Specifically, colon cancer has been linked to one mutation found in approximately 6% of people with Ashkenazi Jewish heritage that replaces isoleucine with lysine at position 1307 in the APC protein.
- (xxiv) Rb1
-
Retinoblastoma 1, or Rb1, acts as a tumor suppressor, therefore, it regulates the cell cycle and prevents cells from dividing at a rapid pace or in an uncontrolled manner. The protein pRB can prevent other proteins from triggering DNA replication. Further, pRB interacts with other proteins to influence cell survival, apoptosis, and differentiation. Mutations in the RB1 gene have been associated with retinoblastoma, a rare type of eye cancer that typically affects young children. About 40% of retinoblastomas are germinal, and thus hereditary, while the other 60% of retinoblastomas are non-germinal, and thus, cannot be passed to the next generation. Bladder cancer, lung cancer, breast cancer, bone cancer, and melanoma have been associated with mutations in the RB1 gene. - (xxv) MEN1
- MEN1 (menin), also referred to multiple endocrine neoplasia I, and acts as a tumor suppressor and is likely involved in several important cell functions. Copying and repairing DNA and apoptosis are functions in which menin plays a role. Menin is also present in the nucleus of many different cell types and appears to be active in all stages of development. Menin also interacts with several transcription factors which bind to specific areas of DNA to help control the expression of the genes. Mutations in MEN1 have been linked to multiple endocrine neoplasia, wherein over 400 mutations in MEN1 have been identified. Familial isolated hyperparathyroidism (FIHP) has also been associated with genetic variation in MEN, as well as sporadic tumors, tumors of the parathyroid gland, pancreatic tumors, and cancerous tumors of the airways in the lungs called bronchial carcinoids.
- (xxvi) VHL
- VHL, or von-Hippel-Lindau tumor suppressor, functions as a part of the VCB-CUL2 complex. This complex targets other proteins to be degraded by the cell when they are no longer required. This degradation removes damaged or unnecessary proteins and helps maintain the normal functions of cells. A protein called hypoxia-inducible factor (HIF) is targeted by the VCB-CUL2 complex to be broken down within cells. HIF controls several important genes involved in cell division and the formation of new blood vessels. VHL protein has also been thought to play a role in other cellular functions, including the regulation of other genes and control of cell division, as well as the formation of extracellular matrix. Mutations in the VHL gene have been linked to von Hippel-Lindau syndrome, wherein over 370 inherited mutations have been identified. The mutations cause an altered or missing VHL protein, leading to a build up of HIF in the cell, signaling the cell to divide abnormally and trigger the production of unnecessary blood vessels. Kidney cancer has also been linked to somatic mutations in VHL. VHL mutations are also linked to hemangioblastoma, Chuvash polycythemia or congenital polycythemia.
- (xxvii) BRCA1
- BRCA1, or
breast cancer 1, early onset, is involved in repairing damaged DNA. BRCA1 protein interacts with several other proteins, including proteins produced by RAD51 and BARD1 genes, to mend breaks in DNA. By helping repair DNA, BRCA1 plays a role in maintaining the stability of a cell's genetic information. It is thought that BRCA1 plays a critical role in embryonic development also. Mutations in BRCA1 are most strongly linked to breast cancer, wherein over 1,000 mutations in the BRCA1 gene have been identified. Most of these over 1,000 mutations lead to the production of an abnormally short BRCA1 protein. Other mutations in BRCA1 change single amino acids in the protein or delete large segments of DNA from the BRCA1 gene. The mutations in BRCA1 often result in a protein that is unable to adequately repair damaged DNA or fix mutations that occur in other genes, allowing cells to divide in an uncontrolled manner, forming a tumor. Mutations in BRCA1 are also associated with an increased risk of fallopian tube cancer, male breast cancer, and pancreatic cancer. - (xxviii) BRCA2
- BRCA2, or breast cancer 2, early onset, interacts with several other proteins, including RAD51 and BARD1, to mend breaks in DNA. By helping repair DNA, BRCA2 plays a role in maintaining the stability of a cell's genetic information. BRCA2 may also help regulate cytokinesis, a step in the cell division process where the cytoplasm divides to form two separate cells. BRCA2 is most strongly associated with breast cancer, wherein over 800 mutations in BRCA2 have been identified. These mutations insert or delete a small number of nucleotides in the gene, which disrupts protein production from one copy of the gene in each cell, resulting in an abnormally small, nonfunctional BRCA2 protein. BRCA2 mutations have also been associated with Fanconi anemia type D1, which results when two faulty copies of the BRCA2 gene are present in each cell. An increased risk of ovarian cancer, prostate cancer, pancreatic cancer, fallopian tube cancer, male breast cancer, and melanoma is associated with genetic variations in BRCA2.
- (xxix) AR
- AR, or androgen receptor, binds an androgen hormone to form an androgen-receptor complex that binds to DNA and regulates the activity of the androgen-responsive genes. Androgen receptors help direct the development of male sexual characteristics, as well as regulating hair growth and sex drive in females. Mutations in the AR gene have been known to cause androgen insensitivity syndrome. Most of the mutations leading to androgen insensitivity syndrome cause changes in single base pairs of DNA, sometimes leading to a shortened version of the AR protein or leading to an abnormal receptor that cannot bind to androgens or to DNA. Other disorders associated with genetic variation in the AR protein include spinal and bulbar muscular atrophy, androgenetic alopecia, breast cancer, prostate cancer, and endometrial cancer in women.
- (xxx) TSG101
- TSG101, or tumor susceptibility gene 101, belongs to a group of inactive homologs of ubiquitin-conjugating enzymes. The protein may play a role in cell growth and differentiation and acts as a negative growth regulator. Genomic stability and cell cycle regulation appear to be linked to the in vivo steady-state expression of TSG101, which acts as a tumor suppressor. Mutations in the TSG101 gene occur in high frequency in breast cancer. (xxxi) Igf1 and Igf2
- Igf1 and Igf2, or insulin-
like growth factor 1 and insulin-like growth factor 2, are structurally and functionally related to insulin but have a much higher growth-promoting activity. Igf2 is thought to play an essential role in growth and development before birth. With regard to the inheritance of Igf2, the copy of the gene inherited from a person's father is the only active copy in most parts of the body. Mutations in Igf1 have been linked to insulin-like growth factor 1 deficiency, an autosomal recessive disorder characterized by growth retardation, sensorineural deafness, and mental retardation. Mutations in Igf2 are associated with Beckwith-Wiedmann syndrome and an increased susceptibility to cancers such as Wilmus tumor, heptaoblastoma, embryonal tumors, Russell-Silver syndrome, breast cancer, prostate cancer, lung cancer, colon cancer, and liver cancer. Normal variations in the Igf2 gene may also be involved in determining the adult height and/or weight of an individual. - (xxxii) Iqf 1R and Iqf 2R
- Igf 1R (insulin-
like growth factor 1 receptor) and Igf 2R (insulin-like growth factor 2 receptor), bind insulin-like growth factors with a high affinity and possess tyrosine kinase activity. Igf 1R plays a critical role in transformation events. Igf 2R is a receptor for both insulin-like growth factor 2 and mannose 6-phosphate. Genetic variance in Igf 1R has been associated with a growth deficiency disorder characterized by intrauterine growth retardation and poor postnatal growth accompanied by increased plasma Igf1. Mutations in Igf 2R are associated with heptocellular carcinoma. - (xxxiii) Bax
- Bax, or BCL-2 associated X protein, encodes for the BCL2 protein. BCL2 family member proteins form hetero-or homodimers and act as anti- or pro-apoptotic regulators that are involved in a variety of cellular activities. The expression of the Bax gene is regulated by the tumor suppressor p53 and has been shown to be involved in p53-mediated apoptosis. Mutations in the Bax gene have been associated with colorectal cancer and t-cell acute lymphoblastic leukemia.
- (xxxiv) Caspase Family (1, 2, 3, 4, 6, 7, 8, 9, 12)
- The caspase family: CASP1, CASP2, CASP3, CASP4, CASP6, CASP7, CASP8, CASP9, and CASP12; are a family of proteases responsible for carrying out the cell death process. The proteases are kept active by proteins on the mitochondrial cell surface from the BCL2 family. When a cell is exposed to cell death signals, such as ischemia, chemotherapy, or radiation, BCL2 function is blocked and caspase activators initiate the cell death cascade. CASP8, caspase 8, is associated with autoimmune lymphoproliferative syndrome, hepatocellular carcinoma, somatic, and lung cancer. CASP12 is associated with the susceptibility to sepsis.
- (xxxv) Kras
- Kras, or v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog, is primarily involved in regulating cell division. K-Ras uses signal transduction to relay signals from the outside of the cell into the cell's nucleus. The signals provided by K-Ras instruct the cell to grow, divide, and differentiate. K-Ras is a GTPase, meaning that it converts GTP to GDP, thus, K-Ras acts like a switch that is turned on and off by the GTP and GDP molecules. The Kras gene belongs to a class of genes known as oncogenes that, when mutated, have the potential to cause normal cells to become cancerous. Mutations in Kras have been linked to Noonan Syndrome, several types of cancers, including pancreatic, lung, and colorectal cancers, cardiofaciocutaneous syndrome, and Costello syndrome.
- (xxxvi) Pten
- Pten, or phosphate and tensin homolog, acts as a tumor suppressor, thus, helps regulate the cycle of cell division by keeping cells from growing and dividing too rapidly or in an uncontrolled way. PTEN modifies other proteins and lipids by removing phosphate groups. The PTEN enzyme acts as part of a chemical pathway that signals the cell to stop dividing and triggers cells to undergo a form of programmed cell death, called apoptosis. PTEN acts to control cell growth so that it does not become irregular or uncontrolled, leading to cancer. Mutations in the PTEN gene have been associated with Cowden syndrome, breast cancer, Bannayan-Riley-Rubalcaba syndrome, Proteus syndrome, and Proteus-like syndrome. These disorders are collectively referred to as PTEN hamartoma tumor syndromes (PHTS).
- (xxxvii) BCRP
- BCRP, or breast cancer resistance protein, has been linked to breast cancer, but its biological significance is largely unknown. BRCP is also known as ABCG2. BRCP is an ATP-binding transport protein that is expressed in several organs, including the liver. BCRP is an ABC transport protein and these transport proteins have been shown to play an important pathophysiological role in several liver diseases.
- (xxxviii) p53
- P53 is a nuclear localized phosphoprotein. P53 is thought to be involved in transcription regulation. Phosphorylation regulates the activity of p53 and the level of p53 is low after mitosis, but increases after G1. Further, p53 may also regulate the initiation of DNA synthesis. Due to the involvement of p53 in both transcription and DNA replication, the various mutants of p53 may also regulate the initiation of DNA synthesis.
- The term “animal,” as used herein, refers to a non-human animal. The animal may be an embryo, a juvenile, or an adult. Suitable animals include vertebrates such as mammals, birds, reptiles, amphibians, and fish. Examples of suitable mammals include without limit rodents, companion animals, livestock, and primates. Non-limiting examples of rodents include mice, rats, hamsters, gerbils, and guinea pigs. Suitable companion animals include but are not limited to cats, dogs, rabbits, hedgehogs, and ferrets. Non-limiting examples of livestock include horses, goats, sheep, swine, cattle, llamas, and alpacas. Suitable primates include, but are not limited to, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys. Non-limiting examples of birds include chickens, turkeys, ducks, and geese. Alternatively, the animal may be an invertebrate such as an insect, a nematode, and the like. Non-limiting examples of insects include Drosophila and mosquitoes. An exemplary animal is a rat. Non-limiting examples of commonly used rat strains suitable for genetic manipulation include Dahl Salt-Sensitive, Fischer 344, Lewis, Long Evans Hooded, Sprague-Dawley and Wistar. In another iteration of the invention, the animal does not comprise a genetically modified mouse. In each of the foregoing iterations of suitable animals for the invention, the animal does not include exogenously introduced, randomly integrated transposon sequences.
- The protein involved in tumor suppression may be endogenous to the animal or it may be exogenous (such as an orthologous tumor suppressor protein). Exogenous proteins may be from any of the animals listed above, as well as from human. The type of genetically modified animal and the source of the protein involved in tumor suppression can and will vary. As an example, the genetically modified animal may be a rat, cat, dog, or pig, and the protein involved in tumor suppression may be human. One of skill in the art will readily appreciate that numerous combinations are possible. In preferred embodiment, the animal is a rat and preferred sequences involved in tumor suppression are listed below. In embodiments in which the genetically modified animal comprises at least one chromosomally integrated sequence encoding an exogenous protein involved in tumor suppression, the exogenous protein is human. In an exemplary embodiment, the genetically modified animal is a rat and the exogenous protein involved in tumor suppression is human.
-
ID Ref Seq. ATM NM_001106821 ATR XR_086326 EGFR NM_031507 ERBB2 NM_017003 ERBB3 NM_017218 ERBB4 NM_021687 Notch1 NM_001105721 Notch2 NM_024358 Notch3 NM_020087 Notch4 NM_001002827 AKT1 NM_033230 AKT2 NM_017093 AKT3 NM_031575 HIF1a, HIF3a NM_024359, NM_022528 Met NM_031517 HRG NM_133428 Bcl2 NM_016993.1 PPAR alpha NM_013196 PPAR gamma NM_013124, NM_001145367, NM_001145366 WT1 (Wilms Tumor) NM_031534 FGF Receptor Family NM_199114, NM_012712, (5 members: 1, 2, 3, 4, 5) NM_001109892, NM_001109893, NM_001109894, NM_001109895, NM_001109896, NM_053429, NM_001109904, NM_199114 (transcript variants) CDKN2a NM_031550 APC NM_012499.1 Rb1 (retinoblastoma) NM_017045 MEN1 NM_019208 VHL NM_052801 BRCA1 NM_012514 BRCA2 NM_031542 AR (Androgen Receptor) NM_012502 TSG101 NM_181628 Igf1 (4 variants) NM_001082477, NM_178866, NM_001082478, NM_001082479 Igf2 (3 variants) NM_031511, NM_001190162, NM_001190163 Igf 1 Receptor NM_052807 Igf 2 Receptor NM_012756 Bax NM_017059 Caspase Family NM_012762, NM_022522, NM_012922, (9 members: 1, 2, 3, 4, NM_053736, NM_031775, 6, 7, 8, 9, 12) NM_022260, NM_022277, NM_031632, NM_130422 Kras NM_031515 PTEN NM_031606 - A further aspect of the present disclosure provides genetically modified cells or cell lines comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression. The genetically modified cell or cell line may be derived from any of the genetically modified animals disclosed herein. Alternatively, the chromosomal sequence involved in tumor suppression may be edited in a cell as detailed below. The disclosure also encompasses a lysate of said cells or cell lines.
- In general, the cells will be eukaryotic cells. Suitable host cells include fungi or yeast, such as Pichia, Saccharomyces, or Schizosaccharomyces; insect cells, such as SF9 cells from Spodoptera frugiperda or S2 cells from Drosophila melanogaster; and animal cells, such as mouse, rat, hamster, non-human primate, or human cells. Exemplary cells are mammalian. The mammalian cells may be primary cells. In general, any primary cell that is sensitive to double strand breaks may be used. The cells may be of a variety of cell types, e.g., fibroblast, myoblast, T or B cell, macrophage, epithelial cell, and so forth.
- When mammalian cell lines are used, the cell line may be any established cell line or a primary cell line that is not yet described. The cell line may be adherent or non-adherent, or the cell line may be grown under conditions that encourage adherent, non-adherent or organotypic growth using standard techniques known to individuals skilled in the art. Non-limiting examples of suitable mammalian cell lines include Chinese hamster ovary (CHO) cells, monkey kidney CVI line transformed by SV40 (COS7), human embryonic kidney line 293, baby hamster kidney cells (BHK), mouse sertoli cells (TM4), monkey kidney cells (CVI-76), African green monkey kidney cells (VERO), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT), rat hepatoma cells (HTC), HIH/3T3 cells, the human U2-OS osteosarcoma cell line, the human A549 cell line, the human K562 cell line, the human HEK293 cell lines, the human HEK293T cell line, and TRI cells. For an extensive list of mammalian cell lines, those of ordinary skill in the art may refer to the American Type Culture Collection catalog (ATCC®, Mamassas, Va.).
- In still other embodiments, the cell may be a stem cell. Suitable stem cells include without limit embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, and unipotent stem cells.
- In general, the genetically modified animal or cell detailed above in sections (I) and (II), respectively, is generated using a zinc finger nuclease-mediated genome editing process. The process for editing a chromosomal sequence comprises: (a) introducing into an embryo or cell at least one nucleic acid encoding a zinc finger nuclease that recognizes a target sequence in the chromosomal sequence and is able to cleave a site in the chromosomal sequence, and, optionally, (i) at least one donor polynucleotide comprising a sequence for integration flanked by an upstream sequence and a downstream sequence that share substantial sequence identity with either side of the cleavage site, or (ii) at least one exchange polynucleotide comprising a sequence that is substantially identical to a portion of the chromosomal sequence at the cleavage site and which further comprises at least one nucleotide change; and (b) culturing the embryo or cell to allow expression of the zinc finger nuclease such that the zinc finger nuclease introduces a double-stranded break into the chromosomal sequence, and wherein the double-stranded break is repaired by (i) a non-homologous end-joining repair process such that an inactivating mutation is introduced into the chromosomal sequence, or (ii) a homology-directed repair process such that the sequence in the donor polynucleotide is integrated into the chromosomal sequence or the sequence in the exchange polynucleotide is exchanged with the portion of the chromosomal sequence.
- Components of the zinc finger nuclease-mediated method are described in more detail below.
- The method comprises, in part, introducing into an embryo or cell at least one nucleic acid encoding a zinc finger nuclease. Typically, a zinc finger nuclease comprises a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease). The DNA binding and cleavage domains are described below. The nucleic acid encoding a zinc finger nuclease may comprise DNA or RNA. For example, the nucleic acid encoding a zinc finger nuclease may comprise mRNA. When the nucleic acid encoding a zinc finger nuclease comprises mRNA, the mRNA molecule may be 5′ capped. Similarly, when the nucleic acid encoding a zinc finger nuclease comprises mRNA, the mRNA molecule may be polyadenylated. An exemplary nucleic acid according to the method is a capped and polyadenylated mRNA molecule encoding a zinc finger nuclease. Methods for capping and polyadenylating mRNA are known in the art.
- (i) Zinc Finger Binding Domain
- Zinc finger binding domains may be engineered to recognize and bind to any nucleic acid sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; and Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc finger binding domain may have a novel binding specificity compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, the disclosures of which are incorporated by reference herein in their entireties. As an example, the algorithm of described in U.S. Pat. No. 6,453,242 may be used to design a zinc finger binding domain to target a preselected sequence. Alternative methods, such as rational design using a nondegenerate recognition code table may also be used to design a zinc finger binding domain to target a specific sequence (see, for example, Biochemistry 2002, 41, 7074-7081).
- A zinc finger binding domain may be designed to recognize a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, or from about 8 to about 19 nucleotides in length. In general, the zinc finger binding domains of the zinc finger nucleases disclosed herein comprise at least three zinc finger recognition regions (i.e., zinc fingers). In one embodiment, the zinc finger binding domain may comprise four zinc finger recognition regions. In another embodiment, the zinc finger binding domain may comprise five zinc finger recognition regions. In still another embodiment, the zinc finger binding domain may comprise six zinc finger recognition regions. A zinc finger binding domain may be designed to bind to any suitable target DNA sequence. See for example, U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, the disclosures of which are incorporated by reference herein in their entireties.
- Exemplary methods of selecting a zinc finger recognition region may include phage display and two-hybrid systems, and are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237, each of which is incorporated by reference herein in its entirety. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in WO 02/077227.
- Zinc finger binding domains and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and are described in detail in U.S. Patent Application Publication Nos. 20050064474 and 20060188987, each incorporated by reference herein in its entirety. Zinc finger recognition regions and/or multi-fingered zinc finger proteins may be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length. See, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, the disclosures of which are incorporated by reference herein in their entireties, for non-limiting examples of linker sequences of six or more amino acids in length. The zinc finger binding domain described herein may include a combination of suitable linkers between the individual zinc fingers of the protein.
- In some embodiments, the zinc finger nuclease may further comprise a nuclear localization signal or sequence (NLS). A NLS is an amino acid sequence which facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome. Nuclear localization signals are known in the art. See, for example, Makkerh et al. (1996) Current Biology 6:1025-1027.
- An exemplary zinc finger DNA binding domain recognizes and binds a sequence having at least about 80% sequence identity with a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8. In other embodiments, the sequence identity may be about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
- (ii) Cleavage Domain
- A zinc finger nuclease also includes a cleavage domain. The cleavage domain portion of the zinc finger nucleases disclosed herein may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388 or www.neb.com. Additional enzymes that cleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). See also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains.
- A cleavage domain also may be derived from an enzyme or portion thereof, as described above, that requires dimerization for cleavage activity. Two zinc finger nucleases may be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease may comprise both monomers to create an active enzyme dimer. As used herein, an “active enzyme dimer” is an enzyme dimer capable of cleaving a nucleic acid molecule. The two cleavage monomers may be derived from the same endonuclease (or functional fragments thereof), or each monomer may be derived from a different endonuclease (or functional fragments thereof).
- When two cleavage monomers are used to form an active enzyme dimer, the recognition sites for the two zinc finger nucleases are preferably disposed such that binding of the two zinc finger nucleases to their respective recognition sites places the cleavage monomers in a spatial orientation to each other that allows the cleavage monomers to form an active enzyme dimer, e.g., by dimerizing. As a result, the near edges of the recognition sites may be separated by about 5 to about 18 nucleotides. For instance, the near edges may be separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides. It will however be understood that any integral number of nucleotides or nucleotide pairs may intervene between two recognition sites (e.g., from about 2 to about 50 nucleotide pairs or more). The near edges of the recognition sites of the zinc finger nucleases, such as for example those described in detail herein, may be separated by 6 nucleotides. In general, the site of cleavage lies between the recognition sites.
- Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme Fok I catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31, 978-31, 982. Thus, a zinc finger nuclease may comprise the cleavage domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Exemplary Type IIS restriction enzymes are described for example in International Publication WO 07/014,275, the disclosure of which is incorporated by reference herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.
- An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimmer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575). Accordingly, for the purposes of the present disclosure, the portion of the Fok I enzyme used in a zinc finger nuclease is considered a cleavage monomer. Thus, for targeted double-stranded cleavage using a Fok I cleavage domain, two zinc finger nucleases, each comprising a Fok I cleavage monomer, may be used to reconstitute an active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage monomers may also be used.
- In certain embodiments, the cleavage domain may comprise one or more engineered cleavage monomers that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 20050064474, 20060188987, and 20080131962, each of which is incorporated by reference herein in its entirety. By way of non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets for influencing dimerization of the Fok I cleavage half-domains. Exemplary engineered cleavage monomers of Fok I that form obligate heterodimers include a pair in which a first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of Fok I and a second cleavage monomer that includes mutations at amino-acid residue positions 486 and 499.
- Thus, in one embodiment, a mutation at amino acid position 490 replaces Glu (E) with Lys (K); a mutation at amino acid residue 538 replaces Iso (I) with Lys (K); a mutation at amino acid residue 486 replaces Gln (Q) with Glu (E); and a mutation at position 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage monomers may be prepared by mutating positions 490 from E to K and 538 from I to K in one cleavage monomer to produce an engineered cleavage monomer designated “E490K:1538K” and by mutating positions 486 from Q to E and 499 from I to L in another cleavage monomer to produce an engineered cleavage monomer designated “Q486E:I499L.” The above described engineered cleavage monomers are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. Engineered cleavage monomers may be prepared using a suitable method, for example, by site-directed mutagenesis of wild-type cleavage monomers (Fok I) as described in U.S. Patent Publication No. 20050064474 (see Example 5).
- The zinc finger nuclease described above may be engineered to introduce a double stranded break at the targeted site of integration. The double stranded break may be at the targeted site of integration, or it may be up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 1000 nucleotides away from the site of integration. In some embodiments, the double stranded break may be up to 1, 2, 3, 4, 5, 10, 15, or 20 nucleotides away from the site of integration. In other embodiments, the double stranded break may be up to 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides away from the site of integration. In yet other embodiments, the double stranded break may be up to 50, 100, or 1000 nucleotides away from the site of integration.
- The method for editing chromosomal sequences involved in tumor suppression may further comprise introducing at least one donor polynucleotide comprising a sequence encoding a protein involved in tumor suppression into the embryo or cell. A donor polynucleotide comprises at least three components: the sequence coding the protein involved in tumor suppression, an upstream sequence, and a downstream sequence. The sequence encoding the protein involved in tumor suppression is flanked by the upstream and downstream sequence, wherein the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome.
- Typically, the donor polynucleotide will be DNA. The donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. An exemplary donor polynucleotide comprising the sequence encoding a protein involved in tumor suppression may be a BAC.
- The sequence of the donor polynucleotide that encodes the protein involved in tumor suppression may include coding (i.e., exon) sequence, as well as intron sequences and upstream regulatory sequences (such as, e.g., a promoter). Depending upon the identity and the source of the orthologous protein involved in tumor suppression, the size of the sequence encoding the protein involved in tumor suppression can and will vary. For example, the sequence encoding the protein involved in tumor suppression may range in size from about 1 kb to about 5,000 kb.
- The donor polynucleotide also comprises upstream and downstream sequences flanking the chromosomal sequence involved in tumor suppression. The upstream and downstream sequences in the donor polynucleotide are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence, as used herein, refers to a nucleic acid sequence that shares sequence similarity with the chromosomal sequence upstream of the targeted site of integration. Similarly, the downstream sequence refers to a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration. The upstream and downstream sequences in the donor polynucleotide may share about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted chromosomal sequence. In other embodiments, the upstream and downstream sequences in the donor polynucleotide may share about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted chromosomal sequence. In an exemplary embodiment, the upstream and downstream sequences in the donor polynucleotide may share about 99% or 100% sequence identity with the targeted chromosomal sequence.
- An upstream or downstream sequence may comprise from about 50 bp to about 2500 bp. In one embodiment, an upstream or downstream sequence may comprise about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. An exemplary upstream or downstream sequence may comprise about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp.
- In some embodiments, the donor polynucleotide may further comprise a marker. Such a marker may make it easy to screen for targeted integrations. Non-limiting examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers.
- One of skill in the art would be able to construct a donor polynucleotide as described herein using well-known standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).
- In the method detailed above for integrating a chromosomal sequence involved in tumor suppression, a double stranded break introduced into the chromosomal sequence by the zinc finger nuclease is repaired, via homologous recombination with the donor polynucleotide, such that the chromosomal sequence involved in tumor suppression is integrated into the chromosome. The presence of a double-stranded break facilitates integration of the sequence into the chromosome. A donor polynucleotide may be physically integrated or, alternatively, the donor polynucleotide may be used as a template for repair of the break, resulting in the introduction of the chromosomal sequence involved in tumor suppression as well as all or part of the upstream and downstream sequences of the donor polynucleotide into the chromosome. Thus, endogenous chromosomal sequence may be converted to the sequence of the donor polynucleotide.
- The method for editing chromosomal sequences involved in tumor suppression may further comprise introducing into the embryo or cell at least one exchange polynucleotide comprising a sequence that is substantially identical to the chromosomal sequence at the site of cleavage and which further comprises at least one specific nucleotide change.
- Typically, the exchange polynucleotide will be DNA. The exchange polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. An exemplary exchange polynucleotide may be a DNA plasmid.
- The sequence in the exchange polynucleotide is substantially identical to a portion of the chromosomal sequence at the site of cleavage. In general, the sequence of the exchange polynucleotide will share enough sequence identity with the chromosomal sequence such that the two sequences may be exchanged by homologous recombination. For example, the sequence in the exchange polynucleotide may have at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with a portion of the chromosomal sequence.
- Importantly, the sequence in the exchange polynucleotide comprises at least one specific nucleotide change with respect to the sequence of the corresponding chromosomal sequence. For example, one nucleotide in a specific codon may be changed to another nucleotide such that the codon codes for a different amino acid. In one embodiment, the sequence in the exchange polynucleotide may comprise one specific nucleotide change such that the encoded protein comprises one amino acid change. In other embodiments, the sequence in the exchange polynucleotide may comprise two, three, four, or more specific nucleotide changes such that the encoded protein comprises one, two, three, four, or more amino acid changes. In still other embodiments, the sequence in the exchange polynucleotide may comprise a three nucleotide deletion or insertion such that the reading frame of the coding reading is not altered (and a functional protein is produced). The expressed protein, however, would comprise a single amino acid deletion or insertion.
- The length of the sequence in the exchange polynucleotide that is substantially identical to a portion of the chromosomal sequence at the site of cleavage can and will vary. In general, the sequence in the exchange polynucleotide may range from about 50 bp to about 10,000 bp in length. In various embodiments, the sequence in the exchange polynucleotide may be about 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 bp in length. In other embodiments, the sequence in the exchange polynucleotide may be about 5500, 6000, 6500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bp in length.
- One of skill in the art would be able to construct an exchange polynucleotide as described herein using well-known standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).
- In the method detailed above for modifying a chromosomal sequence, a double stranded break introduced into the chromosomal sequence by the zinc finger nuclease is repaired, via homologous recombination with the exchange polynucleotide, such that the sequence in the exchange polynucleotide may be exchanged with a portion of the chromosomal sequence. The presence of the double stranded break facilitates homologous recombination and repair of the break. The exchange polynucleotide may be physically integrated or, alternatively, the exchange polynucleotide may be used as a template for repair of the break, resulting in the exchange of the sequence information in the exchange polynucleotide with the sequence information in that portion of the chromosomal sequence. Thus, a portion of the endogenous chromosomal sequence may be converted to the sequence of the exchange polynucleotide. The changed nucleotide(s) may be at or near the site of cleavage. Alternatively, the changed nucleotide(s) may be anywhere in the exchanged sequences. As a consequence of the exchange, however, the chromosomal sequence is modified.
- To mediate zinc finger nuclease genomic editing, at least one nucleic acid molecule encoding a zinc finger nuclease and, optionally, at least one exchange polynucleotide or at least one donor polynucleotide are delivered to the embryo or the cell of interest. Typically, the embryo is a fertilized one-cell stage embryo of the species of interest.
- Suitable methods of introducing the nucleic acids to the embryo or cell include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. In one embodiment, the nucleic acids may be introduced into an embryo by microinjection. The nucleic acids may be microinjected into the nucleus or the cytoplasm of the embryo. In another embodiment, the nucleic acids may be introduced into a cell by nucleofection.
- In embodiments in which both a nucleic acid encoding a zinc finger nuclease and a donor (or exchange) polynucleotide are introduced into an embryo or cell, the ratio of donor (or exchange) polynucleotide to nucleic acid encoding a zinc finger nuclease may range from about 1:10 to about 10:1. In various embodiments, the ratio of donor (or exchange) polynucleotide to nucleic acid encoding a zinc finger nuclease may be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In one embodiment, the ratio may be about 1:1.
- In embodiments in which more than one nucleic acid encoding a zinc finger nuclease and, optionally, more than one donor (or exchange) polynucleotide are introduced into an embryo or cell, the nucleic acids may be introduced simultaneously or sequentially. For example, nucleic acids encoding the zinc finger nucleases, each specific for a distinct recognition sequence, as well as the optional donor (or exchange) polynucleotides, may be introduced at the same time. Alternatively, each nucleic acid encoding a zinc finger nuclease, as well as the optional donor (or exchange) polynucleotides, may be introduced sequentially.
- In some embodiments, the embryo may be derived from an animal comprising at least one edited chromosomal sequence involved in tumor suppression. Thus, an animal comprising 2, 3, 4, or more edited chromosomal sequences involved in tumor suppression may be generated.
- The method of inducing genomic editing with a zinc finger nuclease further comprises culturing the embryo or cell comprising the introduced nucleic acid(s) to allow expression of the zinc finger nuclease. An embryo may be cultured in vitro (e.g., in cell culture). Typically, the embryo is cultured at an appropriate temperature and in appropriate media with the necessary O2/CO2 ratio to allow the expression of the zinc finger nuclease. Suitable non-limiting examples of media include M2, M16, KSOM, BMOC, and HTF media. A skilled artisan will appreciate that culture conditions can and will vary depending on the species of embryo. Routine optimization may be used, in all cases, to determine the best culture conditions for a particular species of embryo. In some cases, a cell line may be derived from an in vitro-cultured embryo (e.g., an embryonic stem cell line).
- Alternatively, an embryo may be cultured in vivo by transferring the embryo into the uterus of a female host. Generally speaking, the female host is from the same or similar species as the embryo. Preferably, the female host is pseudo-pregnant. Methods of preparing pseudo-pregnant female hosts are known in the art. Additionally, methods of transferring an embryo into a female host are known. Culturing an embryo in vivo permits the embryo to develop and may result in a live birth of an animal derived from the embryo. Such an animal would comprise the edited chromosomal sequence involved in tumor suppression in every cell of the body.
- Similarly, cells comprising the introduced nucleic acids may be cultured using standard procedures to allow expression of the zinc finger nuclease. Standard cell culture techniques are described, for example, in Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651; and Lombardo et al (2007) Nat. Biotechnology 25:1298-1306. Those of skill in the art appreciate that methods for culturing cells are known in the art and can and will vary depending on the cell type. Routine optimization may be used, in all cases, to determine the best techniques for a particular cell type.
- Upon expression of the zinc finger nuclease, the chromosomal sequence may be edited. In cases in which the embryo or cell comprises an expressed zinc finger nuclease but no donor (or exchange) polynucleotide, the zinc finger nuclease recognizes, binds, and cleaves the target sequence in the chromosomal sequence of interest. The double-stranded break introduced by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process. Consequently, a deletion, insertion, or point mutation may be introduced in the chromosomal sequence such that the sequence is inactivated.
- In cases in which the embryo or cell comprises an expressed zinc finger nuclease as well as a donor (or exchange) polynucleotide, the zinc finger nuclease recognizes, binds, and cleaves the target sequence in the chromosome. The double-stranded break introduced by the zinc finger nuclease is repaired, via homologous recombination with the donor (or exchange) polynucleotide, such that the sequence in the donor polynucleotide is integrated into the chromosomal sequence (or a portion of the chromosomal sequence is converted to the sequence in the exchange polynucleotide). As a consequence, a sequence may be integrated into the chromosomal sequence (or a portion of the chromosomal sequence may be modified).
- The genetically modified animals disclosed herein may be crossbred to create animals comprising more than one edited chromosomal sequence or to create animals that are homozygous for one or more edited chromosomal sequences. For example, two animals comprising the same edited chromosomal sequence may be crossbred to create an animal homozygous for the edited chromosomal sequence. Alternatively, animals with different edited chromosomal sequences may be crossbred to create an animal comprising both edited chromosomal sequences.
- For example, animal A comprising an inactivated ATM chromosomal sequence may be crossed with animal B comprising a chromosomally integrated sequence encoding a human ATM protein to give rise to a “humanized”
ATM 1 offspring comprising both the inactivated ATM chromosomal sequence and the chromosomally integrated human ATM sequence. Similarly, an animal comprising aninactivated Notch 1 chromosomal sequence may be crossed with an animal comprising a chromosomally integrated sequence encoding thehuman Notch 1 protein to generate “humanized”Notch 1 offspring. Moreover, a humanized ATM animal may be crossed with ahumanized Notch 1 animal to create a humanized ATM/Notch 1 animal. Those of skill in the art will appreciate that many combinations are possible. - In other embodiments, an animal comprising an edited chromosomal sequence disclosed herein may be crossbred to combine the edited chromosomal sequence with other genetic backgrounds. By way of non-limiting example, other genetic backgrounds may include wild-type genetic backgrounds, genetic backgrounds with deletion mutations, genetic backgrounds with other targeted integrations, and genetic backgrounds with non-targeted integrations.
- A further aspect of the present disclosure encompasses methods for using the genetically modified animals. In one embodiment, a genetically modified animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to determine susceptibility to developing tumors. The method comprises exposing the genetically modified animal comprising an inactivated tumor suppressor sequence and a wild-type animal to a carcinogenic agent, and then monitoring the development of tumors. The animal comprising the inactivated tumor suppressor sequence may have an increased risk for tumor formation. Moreover, an animal homozygous for the inactivated tumor suppressor sequence may have increased risk relative to an animal heterozygous for the same inactivated sequence, which in turn may have increased risk relative to a wild-type animal. A similar method may be used to screen for spontaneous tumors, wherein the animals are not exposed to a carcinogenic agent.
- In another embodiment, an animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to evaluate the carcinogenic potential of a test agent. The method comprises contacting the genetically modified animal comprising an inactivated tumor suppressor sequence and a wild-type animal to the test agent, and then monitoring the development of tumors. If the animal comprising an inactivated tumor suppressor sequence has an increased incidence of tumors relative to the wild-type animal, the test agent may be carcinogenic.
- In a further embodiment, an animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to determine the efficacy and/or toxicity of a chemotherapeutic agent or a combination of chemotherapeutic agents. The method comprises inducing tumor formation in genetically modified animal comprising inactivated tumor suppressor sequences, and then comparing the responses of a first group of animals contacted with the chemotherapeutic agent or combination of chemotherapeutic agents to a second group of animal not contacted with the chemotherapeutic agent or combination of chemotherapeutic agents.
- In still another embodiment, an animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used to screen libraries of small molecule drugs for potentially advantageous effects, including enhanced potency as well as reduced untoward effects. The method comprises inducing tumor formation in genetically modified animal comprising inactivated tumor suppressor sequences, and then comparing the responses of a first group of animals contacted with the small molecule drug candidate to a second group of animal not contacted with the small molecule drug candidate.
- In another embodiment, a genetically modified animal comprising an inactivated chromosomal sequence involved with tumor suppression may be used test the ADME/Tox profile of a chemotherapeutic agent or a combination of chemotherapeutic agents. The method is similar to those detailed above, and assessment parameters include damage to DNA, metabolic consequence, and behavioral effects of the chemotherapeutic agent or the combination of chemotherapeutic agents. Behavioral tests include test of learning/memory, anxiety/depression, and sensori-motor functions. Non-limiting examples of behavioral tests suitable for assessing the motor function of rats includes open field locomoter activity assessment, the rotarod test, the grip strength test, the cylinder test, the limb-placement or grid walk test, the vertical pole test, the Inverted grid test, the adhesive removal test, the painted paw or catwalk (gait) tests, the beam traversal test, and the inclined plane test. Non-limiting examples of behavioral tests suitable for assessing the long-term memory function of rats include the elevated plus maze test, the Morris water maze swim test, contextual fear conditioning, the Y-maze test, the T-maze test, the novel object recognition test, the active avoidance test, the passive (inhibitory) avoidance test, the radial arm maze test, the two-choice swim test, the hole board test, the olfactory discrimination (go-no-go) test, and the pre-pulse inhibition test. Non-limiting examples of behavioral tests suitable for assessing the anxiety of rats include the open field locomotion assessment, observations of marble-burying behavior, the elevated plus maze test, the light/dark box test. Non-limiting examples of behavioral tests suitable for assessing the depression of rats includes the forced swim test, the tail suspension test, the hot plate test, the tail suspension test, anhedonia observations, and the novelty suppressed feeding test.
- In yet another embodiment, the genetically modified animals disclosed herein may be used for gene therapy. For example, an animal having a natural mutation in a tumor suppressor gene may genetically modified by editing the chromosomal sequence comprising the natural mutation such that the mutation is corrected. Accordingly, the animal may no longer be susceptible to tumor formation or cancer development.
- Still yet another aspect encompasses a method of generating a cell line or cell lysate using a genetically modified animal comprising an edited chromosomal sequence involved in tumor suppression. An additional other aspect encompasses a method of producing purified biological components using a genetically modified cell or animal comprising an edited chromosomal sequence involved in tumor suppression. Non-limiting examples of biological components include antibodies, receptor proteins, altered tumor suppressor proteins, and the like.
- Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
- The term “chromosomal sequence involved in tumor suppression” refers to a chromosomal sequence which has been identified to contribute to cell cycle maintenance, division of cells, and/or the cell death cycle. Any chromosomal sequence thought to be involved in tumor suppression will work for purposes of the present invention. Exemplary chromosomal sequences involved in tumor suppression include, but are not limited to, ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor 1a), HIF3a (hypoxia-inducible factor 1a), Met (met pronto-oncogene), HRG (histidine-rich glycoprotein), Bc12, PPAR(alpha) (peroxisome proliferator-activated receptor alpha), Ppar(gamma) (peroxisome proliferator-activated receptor gamma), WT1 (Wilmus Tumor 1), FGF1R (fibroblast growth factor 1 receptor), FGF2R (fibroblast growth factor 1 receptor), FGF3R (fibroblast growth factor 3 receptor), FGF4R (fibroblast growth factor 4 receptor), FGF5R (fibroblast growth factor 5 receptor), CDKN2a (cyclin-dependent kinase inhibitor 2A), APC (adenomatous polyposis coli), Rb1 (retinoblastoma 1), MEN1 (multiple endocrine neoplasia1), VHL (von-Hippel-Lindau tumor suppressor), BRCA1 (breast cancer 1), BRCA2 (breast cancer 2), AR (androgen receptor), TSG101(tumor susceptibility gene 101), Igf1 (insulin-like growth factor 1), Igf2 (insulin-like growth factor 2), Igf 1R (insulin-like growth factor 1 receptor), Igf 2R (insulin-like growth factor 2 receptor), Bax (BCL-2 associated X protein), CASP 1 (Caspase 1), CASP 2 (Caspase 2), CASP 3 (Caspase 3), CASP 4(Caspase 4), CASP 6 (Caspase 6), CASP 7 (Caspase 7), CASP 8 (Caspase 8), CASP 9 (Caspase 9), CASP 12 (Caspase 12), Kras (v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog), PTEN (phosphate and tensin homolog), BCRP (breast cancer receptor protein), p53, and combinations thereof.
- The term “a protein encoded by a chromosomal sequence involved in tumor suppression” or “a protein involved in tumor suppression” refers to a protein that has been encoded by a chromosomal sequence which has been identified to contribute to cell cycle maintenance, division of cells, and/or the cell death cycle. Any chromosomal sequence thought to be involved in tumor suppression will work for purposes of the present invention. Exemplary chromosomal sequences involved in tumor suppression include, but are not limited to, ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor 1a), HIF3a (hypoxia-inducible factor 1a), Met (met pronto-oncogene), HRG (histidine-rich glycoprotein), Bc12, PPAR(alpha) (peroxisome proliferator-activated receptor alpha), Ppar(gamma) (peroxisome proliferator-activated receptor gamma), WT1 (Wilmus Tumor 1), FGF1R (fibroblast growth factor 1 receptor), FGF2R (fibroblast growth factor 1 receptor), FGF3R (fibroblast growth factor 3 receptor), FGF4R (fibroblast growth factor 4 receptor), FGF5R (fibroblast growth factor 5 receptor), CDKN2a (cyclin-dependent kinase inhibitor 2A), APC (adenomatous polyposis coli), Rb1 (retinoblastoma 1), MEN1 (multiple endocrine neoplasia1), VHL (von-Hippel-Lindau tumor suppressor), BRCA1 (breast cancer 1), BRCA2 (breast cancer 2), AR (androgen receptor), TSG101(tumor susceptibility gene 101), Igf1 (insulin-like growth factor 1), Igf2 (insulin-like growth factor 2), Igf 1R (insulin-like growth factor 1 receptor), Igf 2R (insulin-like growth factor 2 receptor), Bax (BCL-2 associated X protein), CASP 1 (Caspase 1), CASP 2 (Caspase 2), CASP 3 (Caspase 3), CASP 4(Caspase 4), CASP 6 (Caspase 6), CASP 7(Caspase 7), CASP 8 (Caspase 8), CASP 9 (Caspase 9), CASP 12 (Caspase 12), Kras (v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog), PTEN (phosphate and tensin homolog), BCRP (breast cancer receptor protein), P53, and combinations thereof. Any type of protein involved in tumor suppression will work for purposes of the present invention including, but not limited to, structural proteins, enzyme and catalytic proteins, transport proteins, hormonal proteins, contractile proteins, storage proteins, genetic proteins, defense proteins, and receptor proteins.
- A “gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.
- The terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T.
- The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
- The term “recombination” refers to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, “homologous recombination” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells. This process requires sequence similarity between the two polynucleotides, uses a “donor” or “exchange” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target. Without being bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes. Such specialized homologous recombination often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
- As used herein, the terms “target site” or “target sequence” refer to a nucleic acid sequence that defines a portion of a chromosomal sequence to be edited and to which a zinc finger nuclease is engineered to recognize and bind, provided sufficient conditions for binding exist.
- Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website. With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween. Typically the percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.
- Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between regions that share a degree of sequence identity, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two nucleic acid, or two polypeptide sequences are substantially similar to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more-preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above. As used herein, substantially similar also refers to sequences showing complete identity to a specified DNA or polypeptide sequence. DNA sequences that are substantially similar can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
- Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
- When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a reference nucleic acid sequence, and then by selection of appropriate conditions the probe and the reference sequence selectively hybridize, or bind, to each other to form a duplex molecule. A nucleic acid molecule that is capable of hybridizing selectively to a reference sequence under moderately stringent hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/reference sequence hybridization, where the probe and reference sequence have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press). Conditions for hybridization are well-known to those of skill in the art.
- Hybridization stringency refers to the degree to which hybridization conditions disfavor the formation of hybrids containing mismatched nucleotides, with higher stringency correlated with a lower tolerance for mismatched hybrids. Factors that affect the stringency of hybridization are well-known to those of skill in the art and include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as, for example, formamide and dimethylsulfoxide. As is known to those of skill in the art, hybridization stringency is increased by higher temperatures, lower ionic strength and lower solvent concentrations. With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of the sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. A particular set of hybridization conditions may be selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
- The following examples are included to illustrate the invention.
- The p53 gene was chosen for zinc finger nuclease (ZFN) mediated genome editing. ZFNs were designed, assembled, and validated using strategies and procedures previously described (see Geurts et al. Science (2009) 325:433). ZFN design made use of an archive of pre-validated 1-finger and 2-finger modules. The rat p53 gene region (NM—030989) was scanned for putative zinc finger binding sites to which existing modules could be fused to generate a pair of 4-, 5-, or 6-finger proteins that would bind a 12-18 bp sequence on one strand and a 12-18 bp sequence on the other strand, with about 5-6 bp between the two binding sites.
- Capped, polyadenylated mRNA encoding each pair of ZFNs was produced using known molecular biology techniques. The mRNA was transfected into rat cells. Control cells were transfected with mRNA encoding GFP. Active ZFN pairs were identified by detecting ZFN-induced double strand chromosomal breaks using the Cel-1 nuclease assay. This assay detects alleles of the target locus that deviate from wild type as a result of non-homologous end joining (NHEJ)-mediated imperfect repair of ZFN-induced DNA double strand breaks. PCR amplification of the targeted region from a pool of ZFN-treated cells generates a mixture of WT and mutant amplicons. Melting and reannealing of this mixture results in mismatches forming between heteroduplexes of the WT and mutant alleles. A DNA “bubble” formed at the site of mismatch is cleaved by the surveyor nuclease Cel-1, and the cleavage products can be resolved by gel electrophoresis. This assay revealed that the ZFN pair targeted to bind 5′-atCTGGAGGAAGACtGGAGAAcaagagc-3′ (SEQ ID NO:3; contact sites shown in uppercase) and 5′-atATTCTGGTAAGGAGCCGGgcaagagg-3′ (SEQ ID NO:4) edited the p53 gene.
- Capped, polyadenylated mRNA encoding the active pair of ZFNs was microinjected into fertilized rat embryos using standard procedures (e.g., see Geurts et al. (2009) supra). Control embryos were microinjected with saline or mRNA encoding GFP. The injected embryos were transferred to pseudopregnant female rats to be carried to parturition. Toe/tail of clips of each live born animal was harvested for DNA extraction and analysis using a Cel-1 assay. As shown in
FIG. 1 , about 25% of the experimental animals had an edited p53 gene locus. - To determine that the edited p53 locus was inactivated, Western analyses were performed to confirm that no p53 protein was produced. Cell lysates were prepared from the kidney and liver of a wildtype animal and a p53 knockout animal. As shown on
FIG. 2 , both cytoplasmic and nuclear lysates of the p53 knockout animal were devoid of p53 protein. The levels of actin protein were constant among the wildtype and mutant samples, however. Thus, the p53 edited rat was a p53 knock-out rat. - ZFNs that target and cleave the BCRP gene were identified essentially as described above in Example 1. The rat BCRP gene (NM—1811381) was scanned for putative zinc finger binding sites. ZFNs were assembled and tested essentially as described in Example 1. It was found that the ZFN pair targeted to bind 5′-atGACGTCAAGGAAGAAgtctgcagggt-3′ (SEQ ID NO:5) and 5′-acGGAGATTCTTCGGCTgtaatgttaaa-3′ (SEQ ID NO:6) edited the BCRP gene.
- Rat embryos were microinjected with mRNA encoding the active pair of BCRP ZFNs essentially as described in Examples 1 and 2. The injected embryos were incubated and DNA was extracted from the resultant animals. The targeted region of the BCRP gene was PCR amplified using appropriate primers. The amplified DNA was subcloned into a suitable vector and sequenced using standard methods.
FIG. 3 presents edited BCRP loci in two founder animals. One animal had a 588 bp deletion in exon 7, and the second animal had a 696 bp deletion in exon 7. These deletions disrupt the reading frame of the BCRP coding region. - ZFNs that target and cleave the Pten locus in rats were designed and tested for activity essentially as described above in Example 1. An active pair of ZFNs was identified. The DNA binding sites were 5′-CCCCAGTTTGTGGTCtgcca-3′ (SEQ ID NO:7) and 5′-gcTAAAGGTGAAGATCTA-3′ (SEQ ID NO:8). Capped, polyadenylated mRNA encoding the active pair may be microinjected into rat embryos and the resultant embryos may be analyzed as described in Examples 1 and 2. Accordingly, the Pten locus may be edited to contain a deletion or an insertion such that the coding region is disrupted and no functional protein is made.
- The table below presents the amino acid sequences of helices of the active ZFNs.
-
Name Sequence of Zinc Finger Helices SEQ ID NO: p53 QSGNLAR QSGHLSR DRSALSR QSGNLAR 9 RSDALSR RSDALTQ p53 RSDHLSE TSSDRTK RSDHLSA QSGSLTR 10 RSDVLSE HSNARKT BRCP QSGNLAR QSGNLAR RSDSLST DNASRIR 11 DRSNLTR BRCP QSSDLSR RNDDRKK RREDLIT TSSNLSR 12 QSGHLSR
Claims (34)
1. A genetically modified animal comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression.
2. The genetically modified animal of claim 1 , wherein the edited chromosomal sequence is inactivated, modified, or comprises an integrated sequence.
3. The genetically modified animal of claim 1 , wherein the edited chromosomal sequence is inactivated such that no function tumor suppressor protein is produced.
4. The genetically modified animal of claim 3 , wherein the inactivated chromosomal sequence comprises no exogenously introduced sequence.
5. The genetically modified animal of claim 3 , further comprising at least one chromosomally integrated sequence encoding a functional protein involved in tumor suppression.
6. The genetically modified animal of claim 1 , further comprising a conditional knock-out system for conditional expression of the tumor suppressor protein.
7. The genetically modified animal of claim 1 , wherein the edited chromosomal sequence comprises an integrated reporter sequence.
8. The genetically modified animal of claim 1 , wherein the protein involved in tumor suppression is chosen from ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor 1a), HIF3a (hypoxia-inducible factor 1a), Met (met pronto-oncogene), HRG (histidine-rich glycoprotein), Bc12, PPAR(alpha) (peroxisome proliferator-activated receptor alpha), Ppar(gamma) (peroxisome proliferator-activated receptor gamma), WT1 (Wilmus Tumor 1), FGF1R (fibroblast growth factor 1 receptor), FGF2R (fibroblast growth factor 1 receptor), FGF3R (fibroblast growth factor 3 receptor), FGF4R (fibroblast growth factor 4 receptor), FGF5R (fibroblast growth factor 5 receptor), CDKN2a (cyclin-dependent kinase inhibitor 2A), APC (adenomatous polyposis coli), Rb1 (retinoblastoma 1), MEN1 (multiple endocrine neoplasia1), VHL (von-Hippel-Lindau tumor suppressor), BRCA1 (breast cancer 1), BRCA2 (breast cancer 2), AR (androgen receptor), TSG101 (tumor susceptibility gene 101), Igf1 (insulin-like growth factor 1), Igf2 (insulin-like growth factor 2), Igf 1R (insulin-like growth factor 1 receptor), Igf 2R (insulin-like growth factor 2 receptor), Bax (BCL-2 associated X protein), CASP 1 (Caspase 1), CASP 2 (Caspase 2), CASP 3 (Caspase 3), CASP 4(Caspase 4), CASP 6 (Caspase 6), CASP 7 (Caspase 7), CASP 8 (Caspase 8), CASP 9 (Caspase 9), CASP 12 (Caspase 12), Kras (v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog), PTEN (phosphate and tensin homolog), BCRP (breast cancer receptor protein), P53, and combinations thereof.
9. The genetically modified animal of claim 1 , wherein the protein involved in tumor suppression is chosen from BCRP, p53, PTEN, and combinations thereof.
10. The genetically modified animal of claim 1 , wherein the animal is heterozygous or homozygous for the at least one edited chromosomal sequence.
11. The genetically modified animal of claim 1 , wherein the animal is an embryo, a juvenile, or an adult.
12. The genetically modified animal of claim 1 , wherein the animal is chosen from bovine, canine, equine, feline, ovine, porcine, non-human primate, and rodent.
13. The genetically modified animal of claim 5 , wherein the animal is rat, and the chromosomally integrated sequence encoding a protein involved in tumor suppression is human.
14. A non-human embryo, the embryo comprising at least one RNA molecule encoding a zinc finger nuclease that recognizes a chromosomal sequence encoding a protein involved in tumor suppression, and, optionally, at least one donor polynucleotide comprising a sequence encoding a protein involved in tumor suppression.
15. The non-human embryo of claim 14 , wherein the protein involved in tumor suppression is chosen from ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor 1a), HIF3a (hypoxia-inducible factor 1a), Met (met pronto-oncogene), HRG (histidine-rich glycoprotein), Bc12, PPAR(alpha) (peroxisome proliferator-activated receptor alpha), Ppar(gamma) (peroxisome proliferator-activated receptor gamma), WT1 (Wilmus Tumor 1), FGF1R (fibroblast growth factor 1 receptor), FGF2R (fibroblast growth factor 1 receptor), FGF3R (fibroblast growth factor 3 receptor), FGF4R (fibroblast growth factor 4 receptor), FGF5R (fibroblast growth factor 5 receptor), CDKN2a (cyclin-dependent kinase inhibitor 2A), APC (adenomatous polyposis coli), Rb1 (retinoblastoma 1), MEN1 (multiple endocrine neoplasia1), VHL (von-Hippel-Lindau tumor suppressor), BRCA1 (breast cancer 1), BRCA2 (breast cancer 2), AR (androgen receptor), TSG101(tumor susceptibility gene 101), Igf1 (insulin-like growth factor 1), Igf2 (insulin-like growth factor 2), Igf 1R (insulin-like growth factor 1 receptor), Igf 2R (insulin-like growth factor 2 receptor), Bax (BCL-2 associated X protein), CASP 1 (Caspase 1), CASP 2 (Caspase 2), CASP 3 (Caspase 3), CASP 4(Caspase 4), CASP 6 (Caspase 6), CASP 7 (Caspase 7), CASP 8 (Caspase 8), CASP 9 (Caspase 9), CASP 12 (Caspase 12), Kras (v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog), PTEN (phosphate and tensin homolog), BCRP (breast cancer receptor protein), P53, and combinations thereof.
16. The non-human embryo of claim 14 , wherein the embryo is chosen from bovine, canine, equine, feline, ovine, porcine, non-human primate, and rodent.
17. The non-human embryo of claim 14 , wherein the embryo is rat and the donor polynucleotide comprising a sequence encoding a protein involved in tumor suppression is human.
18. A genetically modified cell, the cell comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppression.
19. The genetically modified cell of claim 18 , wherein the edited chromosomal sequence is inactivated, modified, or comprises an integrated sequence.
20. The genetically modified cell of claim 18 , wherein the edited chromosomal sequence is inactivated such that no function tumor suppressor protein is produced.
21. The genetically modified cell of claim 20 , wherein the inactivated chromosomal sequence comprises no exogenously introduced sequence.
22. The genetically modified cell of claim 20 , further comprising at least one chromosomally integrated sequence encoding a protein involved in tumor suppression.
23. The genetically modified cell of claim 18 , further comprising a conditional knock-out system for conditional expression of the protein involved in tumor suppression.
24. The genetically modified cell of claim 18 , wherein the edited chromosomal sequence comprises an integrated reporter sequence.
25. The genetically modified cell of claim 18 , wherein the cell is heterozygous or homozygous for the at least one edited chromosomal sequence.
26. The genetically modified cell of claim 18 , wherein the cell is of bovine, canine, equine, feline, human, ovine, porcine, non-human primate, or rodent origin.
27. The genetically modified cell of claim 22 , wherein the cell is of rat origin and the chromosomally integrated sequence encoding a protein involved in tumor suppression is human.
28. The genetically modified cell of claim 18 , wherein the protein involved in tumor suppression is ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3 related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch 2, Notch 3, Notch 4, ATK1 (v-akt murine thymoma viral oncogene homolog 1), ATK2 (v-akt murine thymoma viral oncogene homolog 2), ATK3 (v-akt murine thymoma viral oncogene homolog 3), HIF1a (hypoxia-inducible factor 1a), HIF3a (hypoxia-inducible factor 1a), Met (met pronto-oncogene), HRG (histidine-rich glycoprotein), Bc12, PPAR(alpha) (peroxisome proliferator-activated receptor alpha), Ppar(gamma) (peroxisome proliferator-activated receptor gamma), WT1 (Wilmus Tumor 1), FGF1R (fibroblast growth factor 1 receptor), FGF2R (fibroblast growth factor 1 receptor), FGF3R (fibroblast growth factor 3 receptor), FGF4R (fibroblast growth factor 4 receptor), FGF5R (fibroblast growth factor 5 receptor), CDKN2a (cyclin-dependent kinase inhibitor 2A), APC (adenomatous polyposis coli), Rb1 (retinoblastoma 1), MEN1 (multiple endocrine neoplasia1), VHL (von-Hippel-Lindau tumor suppressor), BRCA1 (breast cancer 1), BRCA2 (breast cancer 2), AR (androgen receptor), TSG101 (tumor susceptibility gene 101), Igf1 (insulin-like growth factor 1), Igf2 (insulin-like growth factor 2), Igf 1R (insulin-like growth factor 1 receptor), Igf 2R (insulin-like growth factor 2 receptor), Bax (BCL-2 associated X protein), CASP 1 (Caspase 1), CASP 2 (Caspase 2), CASP 3 (Caspase 3), CASP 4(Caspase 4), CASP 6 (Caspase 6), CASP 7(Caspase 7), CASP 8 (Caspase 8), CASP 9 (Caspase 9), CASP 12 (Caspase 12), Kras (v-Ki-ras2 Kirsten rate sarcoma viral oncogene homolog), PTEN (phosphate and tensin homolog), BCRP (breast cancer receptor protein), and P53.
29. A zinc finger nuclease, the zinc finger nuclease comprising:
a) a zinc finger DNA binding domain that binds a sequence having at least about 80% sequence identity to a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8; and
b) a cleavage domain.
30. The zinc finger nuclease of claim 29 , wherein the sequence identity is at least about 85%, 90%, 95%, or 100%.
31. The zinc finger nuclease of claim 29 , wherein the DNA binding domain comprises at least three zinc finger recognition regions.
32. The zinc finger nuclease of claim 29 , wherein the cleavage domain is a wild-type or an engineered Fok I cleavage domain.
33. A nucleic acid sequence recognized by a zinc finger nuclease, the nucleic acid sequence having at least about 80% sequence identity to a sequence chosen from SEQ ID NOs:3, 4, 5, 6, 7, and 8.
34. A method for assessing the therapeutic effect and/or toxicity of an agent, the method comprising:
a) contacting a first genetically modified animal comprising at least one edited chromosomal sequence encoding a protein involved in tumor suppressor with the agent;
b) measuring a therapeutic and/or toxicity response in the first animal; and
c) comparing the response in (b) to results obtained from a second genetically modified animal comprising the same edited chromosomal sequence encoding a protein involved in tumor suppression, wherein the second animal is not contacted with the agent.
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2010/043167 WO2011011767A1 (en) | 2009-07-24 | 2010-07-23 | Method for genome editing |
US13/386,394 US20120192298A1 (en) | 2009-07-24 | 2010-07-23 | Method for genome editing |
SG2012004131A SG177711A1 (en) | 2009-07-24 | 2010-07-23 | Method for genome editing |
KR1020127004819A KR20120097483A (en) | 2009-07-24 | 2010-07-23 | Method for genome editing |
US12/842,978 US20110023149A1 (en) | 2008-12-04 | 2010-07-23 | Genomic editing of genes involved in tumor suppression in animals |
JP2012521867A JP2013500018A (en) | 2009-07-24 | 2010-07-23 | Methods for genome editing |
AU2010275432A AU2010275432A1 (en) | 2009-07-24 | 2010-07-23 | Method for genome editing |
EP20100803004 EP2456877A4 (en) | 2009-07-24 | 2010-07-23 | Method for genome editing |
CA2767377A CA2767377A1 (en) | 2009-07-24 | 2010-07-23 | Method for genome editing |
IL217409A IL217409A0 (en) | 2009-07-24 | 2012-01-05 | Method for genome editing |
Applications Claiming Priority (16)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US20098508P | 2008-12-04 | 2008-12-04 | |
US20597009P | 2009-01-26 | 2009-01-26 | |
US22841909P | 2009-07-24 | 2009-07-24 | |
US23262009P | 2009-08-10 | 2009-08-10 | |
US24587709P | 2009-09-25 | 2009-09-25 | |
US26369609P | 2009-11-23 | 2009-11-23 | |
US26390409P | 2009-11-24 | 2009-11-24 | |
US12/592,852 US9206404B2 (en) | 2008-12-04 | 2009-12-03 | Method of deleting an IgM gene in an isolated rat cell |
US33600010P | 2010-01-14 | 2010-01-14 | |
US30808910P | 2010-02-25 | 2010-02-25 | |
US30972910P | 2010-03-02 | 2010-03-02 | |
US32369810P | 2010-04-13 | 2010-04-13 | |
US32370210P | 2010-04-13 | 2010-04-13 | |
US32371910P | 2010-04-13 | 2010-04-13 | |
US34328710P | 2010-04-26 | 2010-04-26 | |
US12/842,978 US20110023149A1 (en) | 2008-12-04 | 2010-07-23 | Genomic editing of genes involved in tumor suppression in animals |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/592,852 Continuation-In-Part US9206404B2 (en) | 2008-12-04 | 2009-12-03 | Method of deleting an IgM gene in an isolated rat cell |
US12/842,976 Continuation-In-Part US20120159653A1 (en) | 2008-12-04 | 2010-07-23 | Genomic editing of genes involved in macular degeneration |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/842,980 Continuation-In-Part US20110023150A1 (en) | 2008-12-04 | 2010-07-23 | Genome editing of genes associated with schizophrenia in animals |
Publications (1)
Publication Number | Publication Date |
---|---|
US20110023149A1 true US20110023149A1 (en) | 2011-01-27 |
Family
ID=43498454
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/842,978 Abandoned US20110023149A1 (en) | 2008-12-04 | 2010-07-23 | Genomic editing of genes involved in tumor suppression in animals |
Country Status (1)
Country | Link |
---|---|
US (1) | US20110023149A1 (en) |
Cited By (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110016540A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of genes associated with trinucleotide repeat expansion disorders in animals |
US20110016541A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of sensory-related genes in animals |
US20110016539A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of neurotransmission-related genes in animals |
US20110016543A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genomic editing of genes involved in inflammation |
US20110016542A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Canine genome editing with zinc finger nucleases |
US20110016546A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Porcine genome editing with zinc finger nucleases |
US20110023139A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in cardiovascular disease |
US20110023153A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in alzheimer's disease |
US20110023157A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Equine genome editing with zinc finger nucleases |
US20110023158A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Bovine genome editing with zinc finger nucleases |
US20110023156A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Feline genome editing with zinc finger nucleases |
US20110023146A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in secretase-associated disorders |
US20110023150A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of genes associated with schizophrenia in animals |
US20110023143A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of neurodevelopmental genes in animals |
US20110023144A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in amyotrophyic lateral sclerosis disease |
US20110023147A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of prion disorder-related genes in animals |
US20110023145A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in autism spectrum disorders |
US20110023151A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of abc transporters |
US20110023141A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved with parkinson's disease |
US20110023140A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Rabbit genome editing with zinc finger nucleases |
US20110023148A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of addiction-related genes in animals |
US20110023154A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Silkworm genome editing with zinc finger nucleases |
US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
EP2522726A1 (en) * | 2011-05-12 | 2012-11-14 | Fundació Privada Centre de Regulació Genòmica (CRG) | Zinc finger nucleases for p53 editing |
WO2012152912A1 (en) | 2011-05-12 | 2012-11-15 | Newvectys | Genetically modified pig as a cancer prone model |
US20140245178A1 (en) * | 2013-02-22 | 2014-08-28 | Research In Motion Limited | Communication device and method for profiling and presentation of message threads |
WO2015188094A1 (en) * | 2014-06-06 | 2015-12-10 | President And Fellows Of Harvard College | Methods for targeted modification of genomic dna |
GR1009323B (en) * | 2017-03-23 | 2018-07-02 | Αρχοντης Χαραλαμπου Μιχαηλιδης | Firearm's breech plug with two-way shell ejection |
CN109563508A (en) * | 2016-07-29 | 2019-04-02 | 马克思—普朗克科学促进协会公司 | By fixed point DNA cracking and repair targeting protein diversification in situ |
WO2023208202A1 (en) * | 2022-04-28 | 2023-11-02 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric igf1r |
WO2023236958A1 (en) * | 2022-06-06 | 2023-12-14 | 百奥赛图(北京)医药科技股份有限公司 | Plau and/or plaur gene-modified non-human animal |
Citations (92)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5338678A (en) * | 1989-06-09 | 1994-08-16 | Oncogen, A Limited Partnership | Expression of DNA sequences encoding a thermally stable cytosine deaminase from saccharomyces |
US5356802A (en) * | 1992-04-03 | 1994-10-18 | The Johns Hopkins University | Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease |
US5436150A (en) * | 1992-04-03 | 1995-07-25 | The Johns Hopkins University | Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease |
US5487994A (en) * | 1992-04-03 | 1996-01-30 | The Johns Hopkins University | Insertion and deletion mutants of FokI restriction endonuclease |
US5552311A (en) * | 1993-09-14 | 1996-09-03 | University Of Alabama At Birmingham Research Foundation | Purine nucleoside phosphorylase gene therapy for human malignancy |
US5789538A (en) * | 1995-02-03 | 1998-08-04 | Massachusetts Institute Of Technology | Zinc finger proteins with high affinity new DNA binding specificities |
US5859307A (en) * | 1992-02-04 | 1999-01-12 | Massachusetts Institute Of Technology | Mutant RAG-1 deficient animals having no mature B and T lymphocytes |
US5925523A (en) * | 1996-08-23 | 1999-07-20 | President & Fellows Of Harvard College | Intraction trap assay, reagents and uses thereof |
US6007988A (en) * | 1994-08-20 | 1999-12-28 | Medical Research Council | Binding proteins for recognition of DNA |
US6017896A (en) * | 1993-09-14 | 2000-01-25 | University Of Alabama Research Foundation And Southern Research Institute | Purine nucleoside phosphorylase gene therapy for human malignancy |
US6140466A (en) * | 1994-01-18 | 2000-10-31 | The Scripps Research Institute | Zinc finger protein derivatives and methods therefor |
US6140081A (en) * | 1998-10-16 | 2000-10-31 | The Scripps Research Institute | Zinc finger binding domains for GNN |
US6207150B1 (en) * | 1996-02-09 | 2001-03-27 | Aventis Pharma S.A. | Variants of thymidine kinase, nucleic acids encoding them, and methods of using them |
US6242568B1 (en) * | 1994-01-18 | 2001-06-05 | The Scripps Research Institute | Zinc finger protein derivatives and methods therefor |
US6271436B1 (en) * | 1996-10-11 | 2001-08-07 | The Texas A & M University System | Cells and methods for the generation of transgenic pigs |
US20020004491A1 (en) * | 1999-09-10 | 2002-01-10 | Jiangchun Xu | Compositions and methods for the therapy and diagnosis of ovarian cancer |
US6410248B1 (en) * | 1998-01-30 | 2002-06-25 | Massachusetts Institute Of Technology | General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites |
US20020119570A1 (en) * | 2000-09-25 | 2002-08-29 | Kyonggeun Yoon | Targeted gene correction by single-stranded oligodeoxynucleotides |
US20020127642A1 (en) * | 1996-07-31 | 2002-09-12 | Spurlock Michael E. | Porcine leptin protein, antisense and antibody |
US6453242B1 (en) * | 1999-01-12 | 2002-09-17 | Sangamo Biosciences, Inc. | Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites |
US6479626B1 (en) * | 1998-03-02 | 2002-11-12 | Massachusetts Institute Of Technology | Poly zinc finger proteins with improved linkers |
US6534261B1 (en) * | 1999-01-12 | 2003-03-18 | Sangamo Biosciences, Inc. | Regulation of endogenous gene expression in cells using zinc finger proteins |
US20030083485A1 (en) * | 2001-07-31 | 2003-05-01 | Pfizer Inc. | Novel variants of the human CYP2D6 gene |
US20030232410A1 (en) * | 2002-03-21 | 2003-12-18 | Monika Liljedahl | Methods and compositions for using zinc finger endonucleases to enhance homologous recombination |
US20040019002A1 (en) * | 1999-02-03 | 2004-01-29 | The Children's Medical Center Corporation | Gene repair involving the induction of double-stranded DNA cleavage at a chromosomal target site |
US6706470B2 (en) * | 1999-05-28 | 2004-03-16 | Sangamo Biosciences, Inc. | Gene switches |
US6723893B1 (en) * | 1993-02-26 | 2004-04-20 | Massachusetts Institute Of Technology | Mice having a mutant SOD-1-encoding transgene |
US20050026157A1 (en) * | 2002-09-05 | 2005-02-03 | David Baltimore | Use of chimeric nucleases to stimulate gene targeting |
US20050064474A1 (en) * | 2003-08-08 | 2005-03-24 | Sangamo Biosciences, Inc. | Methods and compositions for targeted cleavage and recombination |
US20050106635A1 (en) * | 2002-03-04 | 2005-05-19 | Maglich Jodi M. | Compositions and methods for regulating thyroid hormone metabolism and cholesterol and lipid metabolism via the nuclear receptor car |
US20050208489A1 (en) * | 2002-01-23 | 2005-09-22 | Dana Carroll | Targeted chromosomal mutagenasis using zinc finger nucleases |
US20050235369A1 (en) * | 2001-03-28 | 2005-10-20 | Yen Choo | Gene regulation II |
US20060063231A1 (en) * | 2004-09-16 | 2006-03-23 | Sangamo Biosciences, Inc. | Compositions and methods for protein production |
US20060188987A1 (en) * | 2003-08-08 | 2006-08-24 | Dmitry Guschin | Targeted deletion of cellular DNA sequences |
US20060199226A1 (en) * | 2005-03-02 | 2006-09-07 | Schiffer Hans H | Functional bioluminescence energy resonance transfer (BRET) assay to screen, identify and characterize receptor tyrosine kinase ligands |
US20060206949A1 (en) * | 2003-01-28 | 2006-09-14 | Sylvain Arnould | Custom-made meganuclease and use thereof |
US20070134796A1 (en) * | 2005-07-26 | 2007-06-14 | Sangamo Biosciences, Inc. | Targeted integration and expression of exogenous nucleic acid sequences |
US20070218528A1 (en) * | 2004-02-05 | 2007-09-20 | Sangamo Biosciences, Inc. | Methods and compositions for targeted cleavage and recombination |
US20070266449A1 (en) * | 2006-05-12 | 2007-11-15 | Zivin Robert A | Generation of animal models |
US20080015164A1 (en) * | 2006-05-19 | 2008-01-17 | Sangamo Biosciences, Inc. | Methods and compositions for inactivation of dihydrofolate reductase |
US20080131962A1 (en) * | 2006-05-25 | 2008-06-05 | Sangamo Biosciences, Inc. | Engineered cleavage half-domains |
US20080159996A1 (en) * | 2006-05-25 | 2008-07-03 | Dale Ando | Methods and compositions for gene inactivation |
US20080200663A1 (en) * | 2004-05-03 | 2008-08-21 | City Of Hope | Novel lentiviral vectors for site-specific gene insertion |
US20080216185A1 (en) * | 2007-01-19 | 2008-09-04 | Invitrogen Corporation | Compositions and Methods for Genetic Manipulation and Monitoring of Cell Lines |
US20080250517A1 (en) * | 1999-03-04 | 2008-10-09 | Alan Colman | Methods |
US20080287651A1 (en) * | 2004-01-13 | 2008-11-20 | Toray Industries, Inc. | Silk Thread Containing Spider Thread Protein and Silk Worm Producing the Silk Thread |
US20080305519A1 (en) * | 2006-02-23 | 2008-12-11 | Qing Lin | Biochemical method for specific protein labeling |
US20090074668A1 (en) * | 2007-09-14 | 2009-03-19 | Farjo Rafal A | Vldlr-/- mouse models and related methods |
US20090111119A1 (en) * | 2007-09-27 | 2009-04-30 | Yannick Doyon | Rapid in vivo identification of biologically active nucleases |
US20090117617A1 (en) * | 2007-10-25 | 2009-05-07 | Sangamo Biosciences, Inc. | Methods and compositions for targeted integration |
US20090137517A1 (en) * | 2006-03-02 | 2009-05-28 | Agency For Science, Technology And Research | Sensitizing a cell to cancer treatment by modulating the activity of a nucleic acid encoding rps27l protein |
US20090215878A1 (en) * | 2008-02-08 | 2009-08-27 | Sangamo Biosciences, Inc. | Treatment of chronic pain with zinc finger proteins |
US20090227029A1 (en) * | 2006-05-10 | 2009-09-10 | Miroslav Radman | Process for Chromosomal Engineering Using a Novel Dna Repair System |
US20090304595A1 (en) * | 2006-05-01 | 2009-12-10 | Aarhus Universitet | Animal model and a method for producing an animal model |
US20100009352A1 (en) * | 2006-05-24 | 2010-01-14 | Gough Albert H | Method for Modeling a Disease |
US20100047805A1 (en) * | 2008-08-22 | 2010-02-25 | Sangamo Biosciences, Inc. | Methods and compositions for targeted single-stranded cleavage and targeted integration |
US20100136710A1 (en) * | 2003-07-02 | 2010-06-03 | Ptc Therapeutics, Inc. | RNA processing protein complexes and uses thereof |
US20100184742A1 (en) * | 2007-06-12 | 2010-07-22 | Manfred Uhr | Polymorphisms in abcb1 associated with a lack of clinical response to medicaments |
US20100218264A1 (en) * | 2008-12-04 | 2010-08-26 | Sangamo Biosciences, Inc. | Genome editing in rats using zinc-finger nucleases |
US20100240090A1 (en) * | 2007-06-15 | 2010-09-23 | Izumi Bio, Inc. | Methods and platforms for drug discovery |
US20100323371A1 (en) * | 2007-07-10 | 2010-12-23 | Immune Disease Institute, Inc. | Stromal interacting molecule knockout mouse and uses thereof |
US20110016539A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of neurotransmission-related genes in animals |
US20110016540A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of genes associated with trinucleotide repeat expansion disorders in animals |
US20110016543A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genomic editing of genes involved in inflammation |
US20110016546A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Porcine genome editing with zinc finger nucleases |
US20110016541A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of sensory-related genes in animals |
US20110016542A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Canine genome editing with zinc finger nucleases |
US20110023147A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of prion disorder-related genes in animals |
US20110023158A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Bovine genome editing with zinc finger nucleases |
US20110023143A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of neurodevelopmental genes in animals |
US20110023150A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of genes associated with schizophrenia in animals |
US20110023159A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Ovine genome editing with zinc finger nucleases |
US20110023139A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in cardiovascular disease |
US20110023140A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Rabbit genome editing with zinc finger nucleases |
US20110023146A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in secretase-associated disorders |
US20110023156A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Feline genome editing with zinc finger nucleases |
US20110023157A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Equine genome editing with zinc finger nucleases |
US20110023153A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in alzheimer's disease |
US20110023145A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in autism spectrum disorders |
US20110023154A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Silkworm genome editing with zinc finger nucleases |
US20110023144A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in amyotrophyic lateral sclerosis disease |
US20110023151A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of abc transporters |
US20110023148A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of addiction-related genes in animals |
US20110023152A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of cognition related genes in animals |
US20110023141A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved with parkinson's disease |
US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
US7956238B2 (en) * | 2006-05-23 | 2011-06-07 | National Taiwan University (An University Of Taiwan, R.O.C.) | Porcine pancreatic amylase gene promoter and transgenic pigs expressing heterologous digestive enzymes |
US20120023599A1 (en) * | 2010-07-23 | 2012-01-26 | Sigma-Aldrich Co. | Genome editing of cytochrome p450 in animals |
US20120030778A1 (en) * | 2008-12-04 | 2012-02-02 | Sigma-Aldrich Co., Llc. | Genomic editing of genes involved with parkinsons disease |
US20120159653A1 (en) * | 2008-12-04 | 2012-06-21 | Sigma-Aldrich Co. | Genomic editing of genes involved in macular degeneration |
US20120159654A1 (en) * | 2008-12-04 | 2012-06-21 | Sigma-Aldrich Co. | Genome editing of genes involved in adme and toxicology in animals |
US20120192298A1 (en) * | 2009-07-24 | 2012-07-26 | Sigma Aldrich Co. Llc | Method for genome editing |
-
2010
- 2010-07-23 US US12/842,978 patent/US20110023149A1/en not_active Abandoned
Patent Citations (97)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5338678A (en) * | 1989-06-09 | 1994-08-16 | Oncogen, A Limited Partnership | Expression of DNA sequences encoding a thermally stable cytosine deaminase from saccharomyces |
US5859307A (en) * | 1992-02-04 | 1999-01-12 | Massachusetts Institute Of Technology | Mutant RAG-1 deficient animals having no mature B and T lymphocytes |
US5487994A (en) * | 1992-04-03 | 1996-01-30 | The Johns Hopkins University | Insertion and deletion mutants of FokI restriction endonuclease |
US5436150A (en) * | 1992-04-03 | 1995-07-25 | The Johns Hopkins University | Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease |
US5356802A (en) * | 1992-04-03 | 1994-10-18 | The Johns Hopkins University | Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease |
US6723893B1 (en) * | 1993-02-26 | 2004-04-20 | Massachusetts Institute Of Technology | Mice having a mutant SOD-1-encoding transgene |
US5552311A (en) * | 1993-09-14 | 1996-09-03 | University Of Alabama At Birmingham Research Foundation | Purine nucleoside phosphorylase gene therapy for human malignancy |
US6017896A (en) * | 1993-09-14 | 2000-01-25 | University Of Alabama Research Foundation And Southern Research Institute | Purine nucleoside phosphorylase gene therapy for human malignancy |
US6140466A (en) * | 1994-01-18 | 2000-10-31 | The Scripps Research Institute | Zinc finger protein derivatives and methods therefor |
US6242568B1 (en) * | 1994-01-18 | 2001-06-05 | The Scripps Research Institute | Zinc finger protein derivatives and methods therefor |
US6007988A (en) * | 1994-08-20 | 1999-12-28 | Medical Research Council | Binding proteins for recognition of DNA |
US6013453A (en) * | 1994-08-20 | 2000-01-11 | Medical Research Council | Binding proteins for recognition of DNA |
US5789538A (en) * | 1995-02-03 | 1998-08-04 | Massachusetts Institute Of Technology | Zinc finger proteins with high affinity new DNA binding specificities |
US6207150B1 (en) * | 1996-02-09 | 2001-03-27 | Aventis Pharma S.A. | Variants of thymidine kinase, nucleic acids encoding them, and methods of using them |
US20020127642A1 (en) * | 1996-07-31 | 2002-09-12 | Spurlock Michael E. | Porcine leptin protein, antisense and antibody |
US6200759B1 (en) * | 1996-08-23 | 2001-03-13 | President And Fellows Of Harvard College | Interaction trap assay, reagents and uses thereof |
US5925523A (en) * | 1996-08-23 | 1999-07-20 | President & Fellows Of Harvard College | Intraction trap assay, reagents and uses thereof |
US6271436B1 (en) * | 1996-10-11 | 2001-08-07 | The Texas A & M University System | Cells and methods for the generation of transgenic pigs |
US6410248B1 (en) * | 1998-01-30 | 2002-06-25 | Massachusetts Institute Of Technology | General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites |
US6479626B1 (en) * | 1998-03-02 | 2002-11-12 | Massachusetts Institute Of Technology | Poly zinc finger proteins with improved linkers |
US6903185B2 (en) * | 1998-03-02 | 2005-06-07 | Massachusetts Institute Of Technology | Poly zinc finger proteins with improved linkers |
US7153949B2 (en) * | 1998-03-02 | 2006-12-26 | Massachusetts Institute Of Technology | Nucleic acid encoding poly-zinc finger proteins with improved linkers |
US6140081A (en) * | 1998-10-16 | 2000-10-31 | The Scripps Research Institute | Zinc finger binding domains for GNN |
US6453242B1 (en) * | 1999-01-12 | 2002-09-17 | Sangamo Biosciences, Inc. | Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites |
US6534261B1 (en) * | 1999-01-12 | 2003-03-18 | Sangamo Biosciences, Inc. | Regulation of endogenous gene expression in cells using zinc finger proteins |
US6607882B1 (en) * | 1999-01-12 | 2003-08-19 | Sangamo Biosciences, Inc. | Regulation of endogenous gene expression in cells using zinc finger proteins |
US20040019002A1 (en) * | 1999-02-03 | 2004-01-29 | The Children's Medical Center Corporation | Gene repair involving the induction of double-stranded DNA cleavage at a chromosomal target site |
US20080250517A1 (en) * | 1999-03-04 | 2008-10-09 | Alan Colman | Methods |
US6706470B2 (en) * | 1999-05-28 | 2004-03-16 | Sangamo Biosciences, Inc. | Gene switches |
US20020004491A1 (en) * | 1999-09-10 | 2002-01-10 | Jiangchun Xu | Compositions and methods for the therapy and diagnosis of ovarian cancer |
US20020119570A1 (en) * | 2000-09-25 | 2002-08-29 | Kyonggeun Yoon | Targeted gene correction by single-stranded oligodeoxynucleotides |
US20050235369A1 (en) * | 2001-03-28 | 2005-10-20 | Yen Choo | Gene regulation II |
US20030083485A1 (en) * | 2001-07-31 | 2003-05-01 | Pfizer Inc. | Novel variants of the human CYP2D6 gene |
US20050208489A1 (en) * | 2002-01-23 | 2005-09-22 | Dana Carroll | Targeted chromosomal mutagenasis using zinc finger nucleases |
US20050106635A1 (en) * | 2002-03-04 | 2005-05-19 | Maglich Jodi M. | Compositions and methods for regulating thyroid hormone metabolism and cholesterol and lipid metabolism via the nuclear receptor car |
US20030232410A1 (en) * | 2002-03-21 | 2003-12-18 | Monika Liljedahl | Methods and compositions for using zinc finger endonucleases to enhance homologous recombination |
US20050026157A1 (en) * | 2002-09-05 | 2005-02-03 | David Baltimore | Use of chimeric nucleases to stimulate gene targeting |
US20060206949A1 (en) * | 2003-01-28 | 2006-09-14 | Sylvain Arnould | Custom-made meganuclease and use thereof |
US20100136710A1 (en) * | 2003-07-02 | 2010-06-03 | Ptc Therapeutics, Inc. | RNA processing protein complexes and uses thereof |
US20050064474A1 (en) * | 2003-08-08 | 2005-03-24 | Sangamo Biosciences, Inc. | Methods and compositions for targeted cleavage and recombination |
US20060188987A1 (en) * | 2003-08-08 | 2006-08-24 | Dmitry Guschin | Targeted deletion of cellular DNA sequences |
US20080287651A1 (en) * | 2004-01-13 | 2008-11-20 | Toray Industries, Inc. | Silk Thread Containing Spider Thread Protein and Silk Worm Producing the Silk Thread |
US20070218528A1 (en) * | 2004-02-05 | 2007-09-20 | Sangamo Biosciences, Inc. | Methods and compositions for targeted cleavage and recombination |
US20080200663A1 (en) * | 2004-05-03 | 2008-08-21 | City Of Hope | Novel lentiviral vectors for site-specific gene insertion |
US20060063231A1 (en) * | 2004-09-16 | 2006-03-23 | Sangamo Biosciences, Inc. | Compositions and methods for protein production |
US20060199226A1 (en) * | 2005-03-02 | 2006-09-07 | Schiffer Hans H | Functional bioluminescence energy resonance transfer (BRET) assay to screen, identify and characterize receptor tyrosine kinase ligands |
US20070134796A1 (en) * | 2005-07-26 | 2007-06-14 | Sangamo Biosciences, Inc. | Targeted integration and expression of exogenous nucleic acid sequences |
US20080305519A1 (en) * | 2006-02-23 | 2008-12-11 | Qing Lin | Biochemical method for specific protein labeling |
US20090137517A1 (en) * | 2006-03-02 | 2009-05-28 | Agency For Science, Technology And Research | Sensitizing a cell to cancer treatment by modulating the activity of a nucleic acid encoding rps27l protein |
US20090304595A1 (en) * | 2006-05-01 | 2009-12-10 | Aarhus Universitet | Animal model and a method for producing an animal model |
US20090227029A1 (en) * | 2006-05-10 | 2009-09-10 | Miroslav Radman | Process for Chromosomal Engineering Using a Novel Dna Repair System |
US20070266449A1 (en) * | 2006-05-12 | 2007-11-15 | Zivin Robert A | Generation of animal models |
US20080015164A1 (en) * | 2006-05-19 | 2008-01-17 | Sangamo Biosciences, Inc. | Methods and compositions for inactivation of dihydrofolate reductase |
US7956238B2 (en) * | 2006-05-23 | 2011-06-07 | National Taiwan University (An University Of Taiwan, R.O.C.) | Porcine pancreatic amylase gene promoter and transgenic pigs expressing heterologous digestive enzymes |
US20100009352A1 (en) * | 2006-05-24 | 2010-01-14 | Gough Albert H | Method for Modeling a Disease |
US20080159996A1 (en) * | 2006-05-25 | 2008-07-03 | Dale Ando | Methods and compositions for gene inactivation |
US20080131962A1 (en) * | 2006-05-25 | 2008-06-05 | Sangamo Biosciences, Inc. | Engineered cleavage half-domains |
US20080216185A1 (en) * | 2007-01-19 | 2008-09-04 | Invitrogen Corporation | Compositions and Methods for Genetic Manipulation and Monitoring of Cell Lines |
US20100184742A1 (en) * | 2007-06-12 | 2010-07-22 | Manfred Uhr | Polymorphisms in abcb1 associated with a lack of clinical response to medicaments |
US20100240090A1 (en) * | 2007-06-15 | 2010-09-23 | Izumi Bio, Inc. | Methods and platforms for drug discovery |
US20100323371A1 (en) * | 2007-07-10 | 2010-12-23 | Immune Disease Institute, Inc. | Stromal interacting molecule knockout mouse and uses thereof |
US20090074668A1 (en) * | 2007-09-14 | 2009-03-19 | Farjo Rafal A | Vldlr-/- mouse models and related methods |
US20090111119A1 (en) * | 2007-09-27 | 2009-04-30 | Yannick Doyon | Rapid in vivo identification of biologically active nucleases |
US20090117617A1 (en) * | 2007-10-25 | 2009-05-07 | Sangamo Biosciences, Inc. | Methods and compositions for targeted integration |
US20090215878A1 (en) * | 2008-02-08 | 2009-08-27 | Sangamo Biosciences, Inc. | Treatment of chronic pain with zinc finger proteins |
US20100047805A1 (en) * | 2008-08-22 | 2010-02-25 | Sangamo Biosciences, Inc. | Methods and compositions for targeted single-stranded cleavage and targeted integration |
US20110016541A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of sensory-related genes in animals |
US20110023157A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Equine genome editing with zinc finger nucleases |
US20110016543A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genomic editing of genes involved in inflammation |
US20110016546A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Porcine genome editing with zinc finger nucleases |
US20110016539A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of neurotransmission-related genes in animals |
US20110016542A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Canine genome editing with zinc finger nucleases |
US20110023147A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of prion disorder-related genes in animals |
US20110023158A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Bovine genome editing with zinc finger nucleases |
US20110023143A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of neurodevelopmental genes in animals |
US20110023150A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of genes associated with schizophrenia in animals |
US20110023159A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Ovine genome editing with zinc finger nucleases |
US20110023139A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in cardiovascular disease |
US20110023140A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Rabbit genome editing with zinc finger nucleases |
US20110023146A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in secretase-associated disorders |
US20110023156A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Feline genome editing with zinc finger nucleases |
US20110016540A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of genes associated with trinucleotide repeat expansion disorders in animals |
US20110023153A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in alzheimer's disease |
US20110023145A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in autism spectrum disorders |
US20110023154A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Silkworm genome editing with zinc finger nucleases |
US20110023144A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in amyotrophyic lateral sclerosis disease |
US20110023151A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of abc transporters |
US20110023148A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of addiction-related genes in animals |
US20110023152A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of cognition related genes in animals |
US20110023141A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved with parkinson's disease |
US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
US20100218264A1 (en) * | 2008-12-04 | 2010-08-26 | Sangamo Biosciences, Inc. | Genome editing in rats using zinc-finger nucleases |
US20120159654A1 (en) * | 2008-12-04 | 2012-06-21 | Sigma-Aldrich Co. | Genome editing of genes involved in adme and toxicology in animals |
US20120030778A1 (en) * | 2008-12-04 | 2012-02-02 | Sigma-Aldrich Co., Llc. | Genomic editing of genes involved with parkinsons disease |
US20120159653A1 (en) * | 2008-12-04 | 2012-06-21 | Sigma-Aldrich Co. | Genomic editing of genes involved in macular degeneration |
US20120192298A1 (en) * | 2009-07-24 | 2012-07-26 | Sigma Aldrich Co. Llc | Method for genome editing |
US20120023599A1 (en) * | 2010-07-23 | 2012-01-26 | Sigma-Aldrich Co. | Genome editing of cytochrome p450 in animals |
Non-Patent Citations (19)
Title |
---|
Beumer (PNAS, Dec. 16, 2008, Vol. 105, No. 50, pg 19821-19826) * |
Bibikova (MCB, Jan. 2001, Vol. 21, No. 1, pg 289-297) * |
Chandrasegaran (Biol. Chem., 1999, Vol. 380, pg 841-848) * |
Donehower (Nature, 1992, Vol. 356, No. 6366, pg 215-221) * |
Doyon (Nature Biotech., June 2008, Vol. 26, No. 6, pg 702-708) * |
Geurts (Science, July 24, 2009, Vol. 325, pg 433-435) * |
Geurts (Science, July 24, 2009, Vol. 325, pg 433-435, Supplemental Material) * |
Jonkers (Nature Genetics, Dec. 2001, Vol. 29, pg 418-425) * |
Mashimo (PLoS ONE, Jan. 2010, Vol. 5, No. 1, e8870, pg 1-7) * |
MGI website description of ApoE, Targeted Allele Description, 2012 * |
NM_030989, 2012 * |
Perez (Nature Biotech., July 2008, Vol. 26, No. 6, pg 808-816) * |
Porteus (Nature Biotech., 2005, Vol. 23, No. 8, pg 967-973) * |
Remy (Transgenic Res. Published online Sept 26, 2009, Vol. 19, pg 363-371) * |
Santiago (PNAS, April 2008, Vol. 105, No. 15, pg 5809-5814) * |
Tong (Nature Sept. 2010, Vol. 467, no. 7312, pg 211-213) * |
Trp53 Targeted Allele Detail MGI Mouse, 2012, pg 1-5 * |
Urnov (Nature Reviews Genetics, Sept. 2010, Vol. 11, pg 636-646) * |
Wu (Cell Mol. Lif Science, 2007, Vol. 64, 2933-2944) * |
Cited By (32)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110023145A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in autism spectrum disorders |
US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
US20110016539A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of neurotransmission-related genes in animals |
US20110016543A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genomic editing of genes involved in inflammation |
US20110016542A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Canine genome editing with zinc finger nucleases |
US20110016546A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Porcine genome editing with zinc finger nucleases |
US20110016540A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of genes associated with trinucleotide repeat expansion disorders in animals |
US20110023153A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in alzheimer's disease |
US20110023157A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Equine genome editing with zinc finger nucleases |
US20110023158A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Bovine genome editing with zinc finger nucleases |
US20110023156A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Feline genome editing with zinc finger nucleases |
US20110023146A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in secretase-associated disorders |
US20110023150A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of genes associated with schizophrenia in animals |
US20110023143A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of neurodevelopmental genes in animals |
US20110023144A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in amyotrophyic lateral sclerosis disease |
US20110023147A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of prion disorder-related genes in animals |
US20110023139A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in cardiovascular disease |
US20110023151A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of abc transporters |
US20110023141A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved with parkinson's disease |
US20110023140A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Rabbit genome editing with zinc finger nucleases |
US20110023148A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of addiction-related genes in animals |
US20110023154A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Silkworm genome editing with zinc finger nucleases |
US20110016541A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of sensory-related genes in animals |
EP2522726A1 (en) * | 2011-05-12 | 2012-11-14 | Fundació Privada Centre de Regulació Genòmica (CRG) | Zinc finger nucleases for p53 editing |
WO2012152912A1 (en) | 2011-05-12 | 2012-11-15 | Newvectys | Genetically modified pig as a cancer prone model |
US20140245178A1 (en) * | 2013-02-22 | 2014-08-28 | Research In Motion Limited | Communication device and method for profiling and presentation of message threads |
WO2015188094A1 (en) * | 2014-06-06 | 2015-12-10 | President And Fellows Of Harvard College | Methods for targeted modification of genomic dna |
CN109563508A (en) * | 2016-07-29 | 2019-04-02 | 马克思—普朗克科学促进协会公司 | By fixed point DNA cracking and repair targeting protein diversification in situ |
US11608570B2 (en) | 2016-07-29 | 2023-03-21 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Targeted in situ protein diversification by site directed DNA cleavage and repair |
GR1009323B (en) * | 2017-03-23 | 2018-07-02 | Αρχοντης Χαραλαμπου Μιχαηλιδης | Firearm's breech plug with two-way shell ejection |
WO2023208202A1 (en) * | 2022-04-28 | 2023-11-02 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric igf1r |
WO2023236958A1 (en) * | 2022-06-06 | 2023-12-14 | 百奥赛图(北京)医药科技股份有限公司 | Plau and/or plaur gene-modified non-human animal |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20110023149A1 (en) | Genomic editing of genes involved in tumor suppression in animals | |
US20110023139A1 (en) | Genomic editing of genes involved in cardiovascular disease | |
US20110023146A1 (en) | Genomic editing of genes involved in secretase-associated disorders | |
US20110016541A1 (en) | Genome editing of sensory-related genes in animals | |
US20120159653A1 (en) | Genomic editing of genes involved in macular degeneration | |
US20110023150A1 (en) | Genome editing of genes associated with schizophrenia in animals | |
EP2558575B1 (en) | Methods for generating endogenously tagged protein | |
US20110023141A1 (en) | Genomic editing of genes involved with parkinson's disease | |
US20120030778A1 (en) | Genomic editing of genes involved with parkinsons disease | |
US20110023145A1 (en) | Genomic editing of genes involved in autism spectrum disorders | |
US20110023144A1 (en) | Genomic editing of genes involved in amyotrophyic lateral sclerosis disease | |
US20110023153A1 (en) | Genomic editing of genes involved in alzheimer's disease | |
US20110016540A1 (en) | Genome editing of genes associated with trinucleotide repeat expansion disorders in animals | |
US20120159654A1 (en) | Genome editing of genes involved in adme and toxicology in animals | |
EP2352369B1 (en) | Genome editing in rats using zinc-finger nucleases | |
US20110023151A1 (en) | Genome editing of abc transporters | |
US20110023148A1 (en) | Genome editing of addiction-related genes in animals | |
US20110023147A1 (en) | Genomic editing of prion disorder-related genes in animals | |
US20110023143A1 (en) | Genomic editing of neurodevelopmental genes in animals | |
US20110023152A1 (en) | Genome editing of cognition related genes in animals | |
US20110023140A1 (en) | Rabbit genome editing with zinc finger nucleases | |
US20120023599A1 (en) | Genome editing of cytochrome p450 in animals | |
Tillotson et al. | Neuronal non-CG methylation is an essential target for MeCP2 function | |
JP6072788B2 (en) | Methods and compositions for altering the cystic fibrosis membrane conductance regulator (CFTR) gene | |
US11957115B2 (en) | Genetically modified mouse expressing human APOE4 and mouse Trem2 p.R47H and methods of use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: SIGMA-ALDRICH CO., MISSOURI Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WEINSTEIN, EDWARD;CUI, XIAOXIA;SIMMONS, PHIL;REEL/FRAME:024923/0509 Effective date: 20100824 |
|
AS | Assignment |
Owner name: SIGMA-ALDRICH CO., LLC, MISSOURI Free format text: MERGER;ASSIGNOR:SIGMA-ALDRICH CO.;REEL/FRAME:026649/0044 Effective date: 20110701 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |